Supporting Information
Text S1: Materials and methods
One BALB/c mouse and one TCRd-/- (BALB/c background) mouse were subcutaneously immunized with
PE/Alum (100 ug per mouse) at the tail base. After 14 days, cells from draining lymph nodes (inguinal) were
stained with Aqua live/dead, Pacific Blue conjugated CD3, H57, CD11b, CD11c,Ter119, Gr-1,F4/80, PE
(100ug/ml), APC-Cy7 conjugated anti-IgD, eFluor605 conjugated B220 1hr on ice. Aqua-,Pacific Blue-,B220+,
PE+ cells or Aqua-,Pacific blue-,B220+, PE- cells were sorted into 5 ul master mix containing 2x reaction mix
(from CellsDirect One-Step qRT-PCR Kit, Invitrogen 11753) and 0.1 ul SUPERase-In RNase Inhibitor (Ambion,
AM2696). The first two and last two wells of each plate were left blank for positive- and negative-controls.
Cells were immediately frozen down at -80 degrees C.
To thaw, plates were spun down at 4 degrees C for 5 minutes at 250xg. Into each well of 5.1 ul cell-lysate,
were added 0.2 ul Superscript III RT-Taq Mix (Invitrogen 11753), 1.3 ul nuclease-free water, and 2.5 ul pre-amp
mix, in TE, containing 200 nM of each DELTAgene-primer pair, 1.2 uM of VHF-1 primer-mix, 1.6 uM of VLF-1
primer-mix, 1 uM of VHR-seq primer-mix, and 0.4 uM of VLR-seq primer-mix. This brought the final volume in
each reaction to 9 ul. To positive-control wells, 1 ul of single-use aliquot splenic total RNA from Balb/C mice
(Agilent 736019) at 9 nmol/ul, was added.
Plates were vortexed 15 s and spun down for 2 minutes at 250xg. Samples were thermocycled 50 degrees C
for 15 minutes, 95 degrees C 2 minutes, and 20x(95 degrees C 15 s, 60 degrees C 4 minutes). Afterward, 4 ul of
Exonuclease-1 mix (New England Biolabs, M0293), containing 20% Exonuclease 1 at 20 units/ul, 1x reaction
buffer, and 70% water, was added, vortexed, and spun down. This was then thermocycled for 30 minutes at
37 degrees C, followed by 15 minutes at 80 degrees C. 14 ul products were diluted 1:5 in TekNova DNA
suspension buffer (TEKnova T0221) and frozen down at -20 degrees C.
After pre-amplification and Exo-1 treatment, diluted samples were thawed. Heavy- and light-chain 4x primer
mixes were prepared, containing either 6.8 uM inVH mix and 4 uM VHR-seq mix, or 8.8 uM inVL mix and 1.6
uM VLR-seq mix, in TE. For each cell-reaction 2.5 ul of each of these were added to separate master-mixes
containing 12.25 ul of nuclease-free water, 2.5 ul HiFi reaction buffer (Invitrogen 11304029), 1 ul 50 mM
MgSO4, 0.5 ul 10 mM dNTP (Invitrogen 18427088), 1.25 ul DMSO (Sigma-Aldrich D9170), and 0.25 ul HiFi
Platinum Taq (Invitrogen 11304029). To these 24 ul mixes, 1 ul of sample was added. After vortexing briefly
and spinning down at 4 degrees C, plates were thermocycled 95 degrees C for 2 minutes, followed by 10x(94
degrees C 1 minute, 62 degrees C 1 minute, 72 degrees C 1.5 minutes), with the temperature 62 degrees C
decreasing by 1 degree Celsius every cycle to 52 degrees C. An additional 30 cycles of 94 degrees C 1 min, 52
degrees C 1 min, 72 degrees C 1.5 min were then performed, followed by a final extension of 72 degrees C for
10 minutes. PCR-products observable on a gel between 400-500 bp were Sanger-sequenced (Sequetech,
Mountain View, CA) using VHR-seq (IgD excluded in order to avoid incoherent signals from IgM/IgD coexpression) or VLR-seq primers (Table S4).
Fluidigm chips were run according to manufacturer’s specifications for single cell EvaGreen quantitative PCR
assays (Fluidigm protocol ADP 30). All 48x48 chips were run on a single Fluidigm Biomark with Data Collection
software v. 3.0.2.
Text S2: Quantitative PCR analysis
Fluidigm chip-readouts were normalized to the median-Ct readouts of their positive controls (each taken from
the same total RNA stock solution). Dilution-curves (comprising total RNA amounts 9000 pg, 900 pg, 90 pg, 20
pg, and 4 pg) showed increasing noise in Ct values above a value of 20. Therefore, 20 was set as the maximumpositive Ct. Cells having GAPDH Ct’s falling above this value were discarded. Purple-colored reactions in Figure
1A of the main text and Figure S4 represent reactions with Ct’s above this maximum value (or those with a
gene-expression value, or –Ct, falling ≥3 below the mean positive gene-expression value for that assay).
We used the amplicon melting curve data to reduce noise in Ct. Comparing the outputted “peak-ratio”
parameter, r, generated by Fluidigm’s Biomark analysis software for each EvaGreen reaction to the high-Tm
(good product fluorescence, g) and low-Tm (bad product fluorescence, b) peaks on individual melting curves,
we found the empirical relation b/g = (1.4-r)/(0.26+r). We used this to correct Ct values by considering each Ct
value as proportional to log2 of the template amount, giving new Ct values Ct’ = Ct + log2(1+b/g). “Perfect”
peak-ratios of 1 or “failed” peak-ratios of 0 were nevertheless left un-corrected or treated as negatives,
respectively. The effect of melting-curve correction was measured both by measuring the average change in
dilution-slopes for Ct-vs.-log2 concentration scatter plots (perfect if equal to -1) and the average
positive/negative accuracy (fraction true-positive or true-negative out of all positive and negative controls).
Before Ct-correction, these values were -0.72±0.28 and 91%±17%, respectively. After Ct-correction, they
improved to -0.75±0.27 and 96%±11%, respectively.1
Figure S1: Clonotype abundances: Antibody sequences clustered by clonal lineage type identified by heavychain CDR3 identity (see Antibody sequence analysis). Evidence for clonal expansion is found among PE+ cells
exclusively.
1
Accuracy calculations assumed all genes in positive control were positive, including those such as RAG1 which were almost
certainly true-negatives. Accuracy percentages should therefore be considered lower-bounds.
Figure S2: Principal components and cell-partitioning. PCA of data depicted in Figure 1 of the main text shows
the first principal component (A), with 45% of total variance, holding the highest values among pre-activation
genes, with IgM and IgD both topping the list. The second principal component (B), with 11% of total variance,
holds the highest values for post-activation genes, with IgG and AID topping the list. Cell-types partition
roughly accordingly (C, dots denote wild-type and circles denote TCRd-/-), with 87 out of 88 PE- B-cells having
a higher value along PC1 and 95 out of 105 PE+ B-cells having a higher value along PC2. On average, this gives
a 94.3% correct partitioning.
Text S3: Antibody sequence analysis
Source code, along with data for this study, are available for download at
http://sourceforge.net/projects/ighanalysis/files/singleCell/
Sequences were analyzed by Smith-Waterman alignments to mouse IMGT reference sequences using software
previously described (Jiang et al 2011). Cells were rejected if they had an average Sanger PHRED-score
(defined as -log10 of the probability of being correct) less than 25, an unidentifiable CDR3 sequence (flanked by
ta(t/c)t(a/t)(c/t)tg(t/c) to the 5’ bound and the best alignment of ta(t/c)tgggg to the 3’ bound as per Kabat et al
1991), or one with ambiguous nucleotides. Of the 368 cells sorted, 193 passed this and the qPCR filter
discussed earlier for further analysis.
For light-chains, the same was done, only replacing the 3’-bound alignment with ttcgg(a/t/c)(g/t/a)(g/c)(g/t/a).
Codon-mutations (non-indels only) were counted, using the CDR3 frame for reference, across the J-region
downstream from the CDR3 region and across the V-region upstream from the CDR3 region for 220 bp.
Mutations from base-calls with PHRED-scores less than 10 were ignored and those with PHRED-scores of at
least 30 were given full weight. PHRED-scores between the two values were used to scale the weights linearly
(eg a mutation with PHRED score of 20 would receive weight ½). Discrete mutation values, such as those
illustrated in Figure 2 of the main text, Figure S4, and Table S1 are rounded from the PHRED-weighted values.
Text S4: Light chain analysis
Because of the far greater potential diversity of heavy-chain rearrangements (due both to the presence of a Dsegment and the greater number of N-nucleotides), heavy-chain sequences provide the best information
regarding the common ancestry of B-cells. Clonotype analysis (Figure S1) was performed by single-linkage
clustering of sequences with identical VH- and JH-segment matches, such that two sequences possessing CDR3
sequences differing by at most 1 amino acid would cluster together. By doing this, we took advantage of the
randomized deletion and insertion of N-nucleotides at the interface between VH, DH, and JH-exons. The total
absence of evidence for clonal expansion among both antigen-negative cell populations helped to confirm that
no systematic cross-contamination had occurred, and among VH-amplicons, no external contamination either.
Nevertheless, the sensitivity of combined pre-amplification and post-amplification on sequence-products
resulted in the failure of 2 out of 8 light-chain negative controls (1 out of 2 for PE-/KO and 1 out of 2 for
PE+/WT). Sequencing the resulting false-positive product showed it bore resemblance to the mixed-signal
obtained from total splenic RNA positive controls.
Eliminating potential light-chain contamination events on the basis of similarity between those belonging to
unique heavy-chains (or heavy-chain clonal lineages) is non-trivial, because of the reduced diversity
mentioned above (light chains lack a D-segment, and many of the light chains sequenced had few or no Nnucleotides at all between their VL and JL exons). Therefore, to establish confidence in each VH- and VLpairing, a stringent statistical test was applied to accept or reject light-chain sequences. For each cell
population, P-values for each light-chain clonal lineage were calculated based on their enrichment within each
heavy-chain lineage using Fisher’s Exact Test:
where LB(A) is the number of cells corresponding to the light-chain (B)/heavy-chain (A) pairing of interest, HA is
the total number of cells possessing the heavy-chain of interest, LB is the total number of cells possessing the
light chain of interest, and N is the total number of cells in the population.
Heavy-chain lineages were iteratively allowed to merge if and only if they would cluster based on a relaxed
criterion (allowing 2 amino acids to differ at their CDR3 region, and their V- and J-segments to belong to the
same exon-families, as opposed to sub-families) and the resulting number of cells in the population
possessing P-values less than 0.03 would be maximally increased. Ties were broken by whichever clustermerger resulted in the least diversity of light chain-lineages within the newly formed heavy-chain lineagecluster. This algorithm would terminate once no VH-lineage merger could keep constant or increase the total
number of cells with light-chain P-values less than 0.03. All light chain sequences having P-values at least 0.03,
or sharing a heavy-chain clonal lineage with a light chain sequence of smaller P-value, were rejected.
The resulting set of light-chain sequences successfully excluded all instances of the detected contaminant, and
merged several existing clonal lineages. As before, evidence for clonal expansion remained exclusively
confined to the PE+ populations – as shown in Figure S5. More generally, this light-chain correction of
antibody clonal lineages did not qualitatively change any of the observed gene-expression/mutation
correlations that incorporated lineage-identity (Figures S6, S7 and Tables S4, S5) or the correspondences
between heavy- and light-chain mutation (Table S1).
Figure S3: Spearman-correlations, in the same manner as main text Figure 2B, were calculated between each
gene-expression and heavy-chain mutation-count belonging to each individual cell measured in the TCRd-/mouse (A). These were depicted alongside wild-type correlations (from main text Figure 2C) by plotting
absolute values along the vertical axis (B) and color-coding according to correlation-sign.
Figure S4: Clustered gene-expression/mutation data. Data are illustrated in the same manner as main text Figure 2,
without stringent filtering of light-chain sequences (described under Light chain analysis). Normalized gene expression
values (red denotes up-regulation and blue denotes down-regulation) were hierarchically-clustered across 193 single
PE+ and PE- B-cells belonging to BALB/c (WT) and TCRd-/- (KO) mice, and plotted alongside mutational content of
antibody heavy- and light-chains expressed by each. For the latter, cells for which light-chains could not be sequenced
are color-coded on the bar-plot in grey.
Figure S5: Clonotype abundances, corrected by light-chain identity. Clonal lineages calculated under the
heavy-chain CDR3-similarity criterion in Figure S1 were re-clustered by incorporating information from
corresponding light-chain data (altered criterion described under Light chain analysis).
Figure S6: Gene-expression correlations calculated by equal-sampling of B-cell clonal lineages (with VLcorrection of lineage-identities). Spearman correlations previously calculated across all cells (Figure 2, Figure
S3) between heavy-chain (IgH) mutations and gene expression were re-calculated by averaging correlations
obtained by iteratively and randomly sampling cells from each lineage (with abundances histogrammed in
Figure 5). Genes which are positive in only a subset of cells from each lineage (so that in at least one instance
of random sampling they are all negative) are excluded from the plot.
Figure S7: Gene-expression correlations calculated by equal-sampling of B-cell clonal lineages (without VLcorrection of lineage-identities). Equal-sampling of mutation/gene-expression pairs applied to clonal lineages
that are assigned by heavy-chain CDR3 similarity alone (as with Figure S1).
Table S1: Contingency tables for heavy/light chain mutation with and without stringent light-chain filtering.
P-values are calculated using a one-tailed Fisher’s Exact Test to quantify the degree to which an un-mutated or
mutated heavy chain predicts an un-mutated or mutated light-chain, respectively.
BALB/c
Gene
P-value
AID
0.001
IGHG1
0.001
BAD
0.009
DOCK8
0.02
IGHM
0.03
IGHG2A/C
0.03
IGHD
0.03
PRDM1
0.04
LTA
0.05
HPRT
0.06
MS4A1
0.06
BCL6
0.07
GAPDH
0.07
CD19
0.09
CD81
0.18
GNAI2
0.19
PRKCD
0.27
FCER2A
0.28
TNFRSF13C
0.31
GUSB
0.31
IGHE
0.33
IGHG2B
0.40
PRKCB
0.43
CD79a
0.45
TNFRSF13B
0.52
HSP90AB1
0.52
HDAC5
0.53
EBI2
0.53
CD40
0.59
CD22
0.66
CLCF1
0.68
IGBP1
0.72
FCGR2B
0.74
MCL1
0.76
IGHA
0.78
IL10
0.80
CDKN1A
0.81
CR2
0.83
IRF4
0.88
Mutation-correlation sign
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
TCRd-/Gene
P-value
GNAI2
0.0002
AID
0.0004
BCL6
0.0004
CD81
0.0005
IGHG2A/C
0.002
MS4A1
0.003
HSP90AB1
0.004
PRKCD
0.005
IGHD
0.006
DOCK8
0.007
CD19
0.007
IGHG1
0.02
HPRT
0.02
GAPDH
0.02
CD79a
0.02
IGBP1
0.03
CD22
0.03
MCL1
0.04
EBI2
0.04
GUSB
0.15
PRKCB
0.15
IGHE
0.16
FCER2A
0.16
BAD
0.19
LTA
0.21
IGHG2B
0.38
CR2
0.56
IL10
0.62
TNFRSF13C
0.63
CLCF1
0.64
IRF4
0.69
FCGR2B
0.71
TNFRSF13B
0.77
IGHM
0.82
CD40
0.82
Mutation-correlation sign
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Table S2: Spearman permutation p-values (two-tailed) derived from the lineage-sampled mutationcorrelation data plotted in Figure 6 (with VL-correction of lineage-identities). P-values are averaged across
105 trials in which cells from each clonal lineage are sampled once. Genes which are positive in only a subset
of cells from each lineage (so that in at least one instance of random sampling they are all negative) are
excluded from the list.
BALB/c
Gene
P-value
IGHG1
0.0006
AID
0.0006
DOCK8
0.01
IGHM
0.01
BAD
0.01
IGHG2A/C
0.01
IGHD
0.02
LTA
0.03
HPRT
0.03
MS4A1
0.03
GAPDH
0.04
BCL6
0.04
PRDM1
0.05
CD19
0.07
GNAI2
0.13
CD81
0.14
PRKCD
0.16
TNFRSF13C
0.19
GUSB
0.22
FCER2A
0.30
IGHE
0.32
HSP90AB1
0.32
IGHG2B
0.34
CD79a
0.40
EBI2
0.45
TNFRSF13B
0.48
HDAC5
0.49
CD40
0.50
PRKCB
0.55
CLCF1
0.59
CD22
0.60
IGBP1
0.67
IGHA
0.72
CDKN1A
0.75
IRF4
0.75
IL10
0.78
MCL1
0.81
FCGR2B
0.83
CR2
0.89
Mutation-correlation sign
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
TCRd-/Gene
P-value
GNAI2
0.0003
AID
0.0006
BCL6
0.0007
CD81
0.0008
IGHG2A/C
0.003
IGHD
0.004
MS4A1
0.005
PRKCD
0.007
HSP90AB1
0.007
DOCK8
0.009
CD19
0.01
IGHG1
0.02
HPRT
0.02
GAPDH
0.03
IGBP1
0.03
EBI2
0.03
CD79a
0.04
CD22
0.04
MCL1
0.04
FCER2A
0.12
IGHE
0.16
GUSB
0.16
PRKCB
0.21
BAD
0.22
LTA
0.24
IGHG2B
0.41
IRF4
0.61
CLCF1
0.64
IL10
0.67
FCGR2B
0.71
CR2
0.71
TNFRSF13C
0.71
IGHM
0.74
TNFRSF13B
0.75
CD40
0.83
Mutation-correlation sign
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Table S3: Spearman permutation p-values (two-tailed) derived from the lineage-sampled mutationcorrelation data plotted in Figure S7 (without VL-correction of lineage-identities). P-values are averaged
across 105 trials in which cells from each clonal lineage are sampled once. Genes which are positive in only a
subset of cells from each lineage (so that in at least one instance of random sampling they are all negative) are
excluded from the list.
VHF-1 mix (outer forward primers)
inVH mix (inner forward primers)
Fraction
Primer name
Sequence
Fraction
Primer name
Sequence
1/24
VHF1_H1-A
ATGAACTTGGGGCTCAGCTT
1/17
inVH1a
AGRTYCAGCTGCARCAGTCT
1/24
VHF1_H1-B
ATGGACTTCGGGCTCAGCTT
1/17
inVH1b
AGGTCCAACTGCAGCAGCC
1/24
VHF1_H1-C
ATGAACTTCGGGCTCAGCTT
1/17
inVH2
TCTGCCTGGTGACWTTCCCA
1/24
VHF1_H1-D
ATGAAGTTGTGGTTAAACTGGGTTTT
1/17
inVH3
GTGCAGCTTCAGGAGTCAG
1/6
VHF1_H2
ATGGAATGSAGCTGGGTCT
1/17
inVH4
GAGGTGAAGCTTCTCGAGTC
1/18
VHF1_H3-A
ATGGACTCCAGGCTCAATTTAGTTTTCCT
1/17
inVH5
GAAGTGAAGCTGGTGGAGTC
1/18
VHF1_H3-B
ATGGCTGTCYTRGBGCTGYTCYTCTG
1/17
inVH6
GAGGAGTCTGGAGGAGGCTT
1/18
VHF1_H3-C
ATGGVTTGGSTGTGGAMCTTGCYATTCCT
1/17
inVH7
CAGTGTGAGGTGAAGCTGGT
1/18
VHF1_H4-A
ATGAAATGCAGCTGGRTYATSTTCTT
1/17
inVH8
CCAGGTTACTCTGAAAGAGTC
1/18
VHF1_H4-B
ATGGRCAGRCTTACWTYYTCATTCCT
1/17
inVH9
ACAGATCCAGTTGGTGCAGT
1/18
VHF1_H4-C
ATGATGGTGTTAAGTCTTCTGTACCT
1/17
inVH10
AGGTGTGCATTGTGAGGTGC
1/18
VHF1_H5-A
ATGGGATGGAGCTRTATCATSYTCTT
1/17
inVH11
GAAGTGCAGCTGTTGGAGAC
1/18
VHF1_H5-B
ATGAAGWTGTGGBTRAACTGGRT
1/17
inVH12
AGCTTCAGGAGTCAGGACC
1/18
VHF1_H5-C
ATGGRATGGASCKKIRTCTTTMTCT
1/17
inVH13
CAGGTGCAGCTTGTAGAGAC
1/24
VHF1_H6-A
ATGAACTTYGGGYTSAGMTTGRTTT
1/17
inVH14
GAGGTTCAGCTGCAGCAGT
1/24
VHF1_H6-B
ATGTACTTGGGACTGAGCTGTGTAT
1/17
inVH15
CAATCCCAGGTTCACCTACAA
1/24
VHF1_H6-C
ATGAGAGTGCTGATTCTTTTGTG
1/17
inVH16
GTGAGGTGCAGCTGGTGGA
1/24
VHF1_H6-D
ATGGATTTTGGGCTGATTTTTTTTATTG
VLF1-mix (outer forward primers)
inVL mix (inner forward primers)
Fraction
Primer name
Sequence
1/22
inVK1
TGATGACCCARACTCCACT
1/8
VLF1-L1
ATGGCCTGGAYTYCWCTYWTMYTCT
1/22
inVK2
GCTTGTGCTCTGGATCCC
1/8
VLF1-K1
ATGRAGWCACAKWCYCAGGTCTTT
1/22
inVK3
CTGCTGCTCTGGGTTCC
1/8
VLF1-K2
ATGGAGACAGACACACTCCTGCTAT
1/22
inVK4
CAGCTTCCTGCTAATCAGTG
1/8
VLF1-K3
ATGGAGWCAGACACACTSCTGYTATGGGT
1/22
inVK5
GTCTCCAGCCACCCTGTC
1/16
VLF1-K4-A
ATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT
1/22
inVK6
TGATGACCCAGTCTCMCAAAT
1/16
VLF1-K4-B
ATGGGCWTCAAGATGRAGTCACAKWYYCWGG
1/22
inVK7
GCCTGTGCAGACATTGTGAT
1/24
VLF1-K5-A
ATGAGTGTGCYCACTCAGGTCCTGGSGTT
1/22
inVK8
CCTGTGGGGACATTGTGATG
1/24
VLF1-K5-B
ATGTGGGGAYCGKTTTYAMMCTTTTCAATTG
1/22
inVK9
CWTCTTGTTGCTCTGGTTTC
1/24
VLF1-K5-C
ATGGAAGCCCCAGCTCAGCTTCTCTTCC
1/22
inVK10
CCAGATGTGATATCCAGATG
1/32
VLF1-K6-A
ATGAGIMMKTCIMTTCAITTCYTGGG
1/22
inVK11
GCCAGATGTGATGTYCAAATG
1/32
VLF1-K6-B
ATGAKGTHCYCIGCTCAGYTYCTIRG
1/22
inVK12
CTGCTGCTGTGGCTTACA
1/32
VLF1-K6-C
ATGGTRTCCWCASCTCAGTTCCTTG
1/22
inVK13
CCTTCTCAACTTCTGCTCT
1/32
VLF1-K6-D
ATGTATATATGTTTGTTGTCTATTTCT
1/22
inVK14
MAGATGACCCAGTCTCCATC
1/32
VLF1-K7-A
ATGAAGTTGCCTGTTAGGCTGTTGGTGCT
1/22
inVK15
TGAGATGTGACATCCAGATGA
1/32
VLF1-K7-B
ATGGATTTWCARGTGCAGATTWTCAGCTT
1/22
inVK16
CCAGTGTGATGTCCAGATAAC
1/32
VLF1-K7-C
ATGGTYCTYATVTCCTTGCTGTTCTGG
1/22
inVK17
ACAACTGTGACCCAGTCTCC
1/32
VLF1-K7-D
ATGGTYCTYATVTTRCTGCTGCTATGG
1/22
inVK18
ACACAGGCTCCAGCTTCTCT
1/22
inVK19
GTGCTCAGTGTGACATCCAG
1/22
inVL1
TCCCAGGCTGTTGTGACTC
1/22
inVL2
CAACTTGTGCTCACTCAGTC
1/22
inVL3
ACTCAGCCAAGCTCTGTG
VHR-seq mix (reverse primers)
VLR-seq mix (reverse primers)
Fraction
Primer name
Sequence
Fraction
Primer name
Sequence
1/5
VHR-IgA
GTGCCGRAAGGGAAGTAATC
1/2
VLR-IgL
CACACCAGTGTGGCYTTGTT
1/5
VHR-IgD
CAGCCCAGGTTTATCTTTTCA
1/4
VLR-IgK-A
CAWGAAGCACACGACTGAGG
1/5
VHR-IgG
GGGAARTAVCCCTTGACCAG
1/4
VLR-IgK-B
CAGGAAACACACGATTGAGG
1/5
VHR-IgM
CCATGGCCACCAGATTCTT
1/5
VHR-IgE
ACCAGGTCACAGTCACAGGA
Table S4: Sequencing-amplicon primer-sets. Outer forward primer-sets VHF-1 and VLF-1 (adapted from
Novagen cat. 69831-3) were used during pre-amplification of sequencing-amplicons. Inner forward primersets inVH and inVL (adapted from Rohatgi et al 2008) were used independently during post-amplification
(prior to sequencing). Reverse primer-sets VHR-seq and VLR-seq targeting constant-regions were used during
both steps. Fractions in left-most columns denote proportions of primers in mixes (all add to 1).
Name
AID
BAD
BCL6
CD19
CD22
CD40
CD5
CD79a
CD81
CDKN1A
CLCF1
CR2
DOCK8
FCAMR
FCER2A
FCGR2B
GAPDH
GNAI2
EBI2
GUSB
HDAC5
HPRT
HSP90AB1
IGBP1
IGHD
IGHE
IGHG1
IGHG2A/C
IGHG2B
IL10
IL12B
IRF4
IGHA
IGHM
LTA
MCL1
MS4A1
PIK3CD
PRDM1
PRKCB
PRKCD
PTPRC
RAG1
SLA2
TNFRSF13B
TNFRSF13C
TNFRSF8
TNFSF8
Forward
CCTGGGAAGGGCTACATGAAA
GCAGCCACCAACAGTCATCA
GGGGAAACCCAGTCAGAGTA
CCATCGAGAGGCACGTGAA
CAGCAGGGGCTTCAGGAAAA
CTATGGGGCTGCTTGTTGAC
AGCAGTGCTTCCAGAAAACAAC
AAGAACCACAGGGGCTTGTA
GCTGTACCTGGAACTGGGAA
GAACATCTCAGGGCCGAAAAC
AGCATCAACTCCGCAGCTTA
CCATCTGGACTAAGAAGCCAGTA
GTACAAGGAACCCGCAATCA
CAAGCCAGCTTTCAGTAGCA
AGGTGGCAAAGCTGTGGATA
CTCACGGACTTTGTGCCATA
AGACGGCCGCATCTTCTT
TTTGGCCGCTCACGAGAATA
CAAACACGGACTGCCACAAC
AGTATGGAGCAGACGCAATCC
CCCCATCAGCCAGAAGATGTA
CAGTACAGCCCCAAAATGGTTA
CCTCCGCGAGTTGATCTCTAA
CCTCCAATGAAACCCTTCATCC
ATCAGCTGGGAGCCAAAGAA
CCTGAACATGAGCACTGTGAAC
TGGAACTCTGGATCCCTGT
AACTCTGGATCCCTGTCCAGT
CACACCTTCCCAGCTCTCC
AAAGGACCAGCTGGACAACA
ATCGTTTTGCTGGTGTCTCC
TCCCCATTGAGCCAAGCATA
TGGGGAAAGAGTGGGAAGGATA
AGGGTATCAGAACCTTCCCAAC
ACTCCCTCAGAAGCACTTGAC
AAACGGGACTGGCTTGTCA
GTGACAGCTGGTATTGTGGAGAA
CTTATTGCGTGTCAGCAACC
CGTTCGGTCAGCTCTCCAA
CACATCGACAGAGAGGTTCTCA
CCTCAACAAGCAAGGCTACA
TGATGAGGGCAGACTGTTCC
TCTCTGTGGCATCGAGTGTTA
ACCTGTGACTGTGCCAACA
GGTGGAAGTCAGGTCAGACAA
TTCGACCCTCTGGTGAGAAAC
CAGCACCTCAGAAAACAGCAA
ATCCAAGAAGTCATGGGCCTAC
Reverse
CGCAAGTCATCGACTTCGTAC
GTACGAACTGTGGCGACTCC
CTCAGAGAAACGGCAGTCAC
ACCACTGGGACTATCCATCCA
TCTGAGAGAGGGGTCCTCCTA
TCGTGGAGGTACTGTTTGTCA
CTCTGAACCTTGGGCTGGAA
CATGTCCAGGAAGGGCCTA
AGCTCCCACAGCAATGAGAA
TCTGCGCTTGGAGTGATAGAA
CAGTCGAGGAGGATTGAAGTCA
AGCTGCCTGTATGACTTCCA
CACCGAAACACTGGCCATAA
AGTGTGGCTTTCTCCTTCCA
GGAGCCAGTTCTTGGGACATA
CATGAGTCCCAGCAGCAA
TTCACACCGACCTTCACCAT
TCTGTGCAATGCGCTCCA
GACGTTGCCAGTGGGGTA
ACAGCCTTCTGGTACTCCTCA
TCTCATTCCACACCGTGTCA
AGTCTGGCCTGTATCCAACA
CCGTCAGGCTCTCATATCGAA
GTTGCCAGGCTTGGATAACC
GTGACCTGGAGGACCATTGTA
TCACTTGGCTGGTGGTGAC
GAGCTGCTCAGAGTGTAGAG
TGAGCTGCTGAGGGTGTAGA
TGCTGGAGGGGACAGTCA
TAAGGCTTGGCAACCCAAGTA
GGAGTCCAGTCCACCTCTAC
CGAGGATGTCCCGGTAATACA
ACTGGCTGCTCATGGTGTAC
ATGCTCTTGGGAGACAGCAA
GAGTTCTGCTTGCTGGGGTA
CCGCCTTCTAGGTCCTGTAC
CCAGCTGACAGCAGAACCA
TCCTTGGTGGATACCTGGAA
TGCAGGTCTGGCACTTGAAA
ACAAGCCATTGGGGTCCATA
GCGGCCGATAATCTTGTCA
TCGGGCATCTTTGATGGGAA
AGGCAGCCATGTTGGCTAA
TCGCAGGTGCTTCCAGAAA
CTGGTCGCTACTTAGCCTCAA
AGCTGTCCCAGGCTCCA
TGCTGGTCCAGGCATCTAC
ATGGTGCCATCTTCGTTCCA
RefSeq
NM_009645.2
NM_007522.2
NM_009744.3
NM_009844.2
NM_001043317.2
NM_011611.2
NM_007650.3
NM_007655.3
NM_133655.2
NM_007669.4
NM_019952.3
NM_007758.2
NM_028785.3
NM_001170632.1
NM_013517.4
NM_010187.2
NM_008084.2
NM_008138.4
NM_183031.2
NM_010368.1
NM_010412.3
NM_013556.2
NM_008302.3
NM_008784.3
IMGT IgD-CH1
IMGT IgE-CH1
IMGT IgG1-CH1
IMGT IgG2A/C-CH1
IMGT IgG2B-CH1
NM_010548.2
NM_008352.2
NM_013674.1
IMGT IgA-CH1
IMGT IgM-CH1
NM_010735.2
NM_008562.3
NM_007641.5
NM_008840.3
NM_007548.3
NM_008855.2
NM_011103.2
NM_011210.3
NM_009019.2
NM_029983.5
NM_021349.1
NM_028075.2
NM_009401.2
NM_009403.3
Table S5: EvaGreen PCR assay primer-sets. Assays were designed by Fluidigm DELTAgene. Antibody isotypespecific assays (IgHA, IgHD, IgHE, IgHG, and IgHM) were designed against portions of consensus sequences of
mouse CH1 regions available on the IMGT database that were mutually exclusive to sequencing amplicons.
IgHG2A/C primers were designed against both IgHG2A and IgHG2C subclasses, due to homology. Only those
genes producing positive signal in at least one cell are shown in Figure 1 of the main text.
Name
Biochem. Features
AID
BAD
BCL6
CD19
CD22
CD40
CD5
CD79a
CD81
CDKN1A
CLCF1
CR2
DOCK8
FCAMR
FCER2A
FCGR2B
GAPDH
GNAI2
EBI2
GUSB
HDAC5
HPRT
HSP90AB1
IGBP1
IGHD
IGHE
IGHG1
IGHG2A/C
IGHG2B
IL10
IL12B
IRF4
IGHA
IGHM
LTA
MCL1
MS4A1
PIK3CD
PRDM1
PRKCB
PRKCD
PTPRC
RAG1
SLA2
TNFRSF13B
TNFRSF13C
TNFRSF8
TNFSF8
Cytidine deamination
Somatic mutation/isotype switching
Protein-binding
Positive regulation of apoptosis
DNA-binding
B cell differentiation/germinal center formation
Integral membrane
B cell receptor signaling pathway
Protein-binding
Cell surface receptor linked signaling pathway
Enzyme-binding
Positive regulation of B cell proliferation
Glycoprotein-binding
T cell costimulation
Protein-binding
B cell activation
Protein-binding
Positive regulation of B cell proliferation
Cyclin-binding
DNA damage response/regulation of cell cycle
Cytokine activity
B cell differentiation
Homodimerization
B cell activation/complement activation
GTP binding
Antibody signal transduction
IgA/IgM-binding
Antibody heavy-chain receptor/signal transduction
IgE-binding
Antibody heavy-chain receptor/signal transduction
IgG-binding
Antibody heavy-chain receptor/signal transduction
Dehydrogenase
Cell metabolism
GTP binding
B cell motility
G-protein coupled receptor
B cell migration
Beta-glucuronidase
Carbohydrate metabolism
Histone deacetylase
B cell activation/chromatin modification/cell cycle
Guanine phosphoribosyltransferase
GMP salvage/nucleoside metabolism
Unfolded-protein binding
Protein folding/response to stress
Mitogen-activated
B cell activation/negative regulation of apoptosis
Antigen-binding
Antibody heavy-chain/antigen-recognition
Antigen-binding
Antibody heavy-chain/antigen-recognition
Antigen-binding
Antibody heavy-chain/antigen-recognition
Antigen-binding
Antibody heavy-chain/antigen-recognition
Antigen-binding
Antibody heavy-chain/antigen-recognition
Cytokine activity
Negative regulation of B cell proliferation
Cytokine activity
B cell activation and proliferation
DNA-binding
Histone acetylation/plasma cell differentiation
Antigen-binding
Antibody heavy-chain/antigen-recognition
Antigen-binding
Antibody heavy-chain/antigen-recognition
Cytokine activity
B cell migration
BH domain-binding
Negative regulation of apoptosis
Membrane-spanning
B cell activation
1-PI-3-kinase
B cell activation
DNA-binding
Plasma cell differentiation
Calcium-channel regulator
B cell activation
Enzyme-binding
B cell proliferation/positive regulation of apoptosis
Phosphatase
B cell differentiation/positive regulation of apoptosis
DNA-binding
B cell differentiation/VDJ recombination
SH3/SH2 adaptor activity
Negative-regulation of antibody-signaling
Receptor activity
Negative regulation of B cell proliferation
Receptor activity
Positive regulation of B cell proliferation
Receptor activity
Positive regulation of NFkB TF activity
Cytokine activity
Signal transduction during B cell activation
Role in B cell biology
Chromosomal location
Chr6: 122553809..122564180
Chr19:6941855..6951893
Chr16: 23965052..23988612
Chr7: 126408448..126414870
Chr7: 30865404..30880342
Chr2: 165055636..165071654
Chr19: 10718143..10738974
Chr7: 24897511..24902197
Chr7: 143052750..143067930
Chr17: 29090986..29100722
Chr19: 4214392..4222615
Chr1: 195136811..195176715
Chr19: 24999529..25202432
Chr1: 130800902..130814740
Chr8: 3681737..3694174
Chr1: 170960558..170976071
Chr6: 125161852..125165583
Chr9: 107614138..107635342
Chr14: 121952331..121965193
Chr5: 129989021..130002828
Chr11: 102195747..102230172
ChrX: 52988078..53021660
Chr17: 45567778..45573261
ChrX: 100494291..100516125
Chr12: 113407535..113416324
Chr12: 113269263..113273248
Chr12: 113326544..113330523
Chr12: 110806667..110813558
Chr12: 113304314..113307933
Chr1: 131019845..131024970
Chr11: 44400063..44414017
Chr13: 30749258..30766927
Chr12: 113256204..113260236
Chr12: 113418826..113422730
Chr17: 35203165..35205351
Chr3: 95658721..95663179
Chr19: 11250603..11266151
Chr4: 149649168..149701629
Chr10: 44437175..44458687
Chr7: 122289125..122634402
Chr14: 30595354..30626208
Chr1: 138062859..138175305
Chr2: 101638252..101649532
Chr2: 156872922..156887078
Chr11: 61140835..61147642
Chr15: 82221744..82224336
Chr4: 145268976..145315147
Chr4: 63832824..63861284
Table S6: Summary of gene biochemical features, roles in B cell biology, and chromosomal locations. See
main text for references.
References
Jiang N, Weinstein JA, Penland L, White RA 3rd, Fisher DS, Quake SR (2011) Determinism and stochasticity
during maturation of the zebrafish antibody repertoire. Proc Natl Acad Sci U S A108:5348-53.
Kabat E A, Wu T T, Perry H M, Gottesman K S, Foeller C (1991) Sequences of Proteins of Immunological
Interest. Vol. 1. 5th ed, 1991.
Rohatgi S, Ganju P, Sehgal D (2008) Systematic design and testing of nested (RT-)PCR primers for specific
amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B
cells. J Immunol Methods. 339:205-19.