SUPPLEMENTARY MATERIALS AND METHODS
Moldt et al. Comparative genomic integration profiling of Sleeping Beauty transposons mobilized with
high efficacy from integrase-defective lentiviral vectors in primary human cells
Linear amplification-mediated PCR (LAM-PCR) for the amplification of the vector-genomic DNA junctions
Three µg of purified genomic DNA was randomly fragmented using the Covaris S2 sonication devise with
the following settings: 2 times 45 sec; duty cycle: 20%; intensity: 5; cycle/burst: 200. The fragmented DNA
was ethanol precipitated and dissolved in 30 µl TE buffer. 500 ng DNA was used as input for linear
amplification-mediated PCR (LAM-PCR) [1] in a 50 µl reaction with 1 pmol LAM-SB/L-50-Bio or LAM-LV-50Bio with the following cycling conditions: 94oC, 3 min; 50 cycles of: 94oC, 30s; ramping to 54oC, 1 oC/s; 54oC,
30s, 72oC 1min. Next, 2.5 U of fresh Taq polymerase was given to the reaction and the program above was
repeated. The products were then bound to streptavidin-coated magnetic beads overnight using the
Dynabeads kilobase BINDER kit (Invitrogen) as described [1]. All subsequent steps were performed on the
magnetic bead-bound DNA. Repeated washing steps with 100µl H2O followed each reaction. Second strand
synthesis was performed with Klenow using Hexanucleotide mix (Roche) and 0.25mM dNTP in a 20 µl
reaction volume at 37oC for 40min. End repair and 5’ phosphorylation of the DNA was performed using the
End-it DNA End-Repair kit (Epicentre) as recommended. Next, 2.5U exo- Klenow fragment (NEB) was used
to add a singe “A” nucleotide to the 3’ ends of the fragments at 37 oC, for 30 min. Ligation of the fragments
to 50pmol linkers with “T”-overhangs were performed at 4oC overnight. After final washing steps with
water, the beads were resuspended in 20ul of TE buffer. 2 µl of the bead suspension were used for the first
round of nested exponential PCR amplification steps with the primers Linker and LAM-SB-L-20 or LAM-LV34, using the following conditions: 94oC, 2 min; 30 cycles of: 94oC, 30s, ramping to 55oC, 1oC/s; 55oC, 20s;
72oC, 30s; 72oC. 1 µl of the first PCR was used in the nested PCR round with the primers Nested and LAMLV-BC-OVH or LAM-SBIII-BC-OVH (where BC stands for different four-letter barcodes) using the following
program: 94oC, 2 min; 30 cycles of: 94oC, 30s, ramping to 51oC, 1oC/s; 51oC, 20s; 72oC, 30s. 1µl of the
second PCR was used to attach the linker sequences compatible with the Illumina flow-cells to the
fragments using Phusion polymerase (Finnzymes), with the primers Illumina-1 and Illumina-2 in a 50 µl
reaction volume as follows: 98oC, 30 sec; 15 cycles of: 98oC, 10s; 65oC, 20s; 72oC, 30s. If necessary, the latter
PCR was scaled up to 100 µl and the PCR products of 100-500 bp range were isolated from 1.2% agarose
gels and purified using Qiaquick Gel Extraction kit (Qiagen).
The linker with 5’ “T” overhang was obtained by annealing the following oligos: Sonic TA Link(+) and Sonic
Link (-) amino
Primer Name
Sequence (5’-3’), caps stand for barcodes
LAM-SB/L-50-Bio
Biotin-agttttaatgactccaacttaagtg
LAM-LV-50-Bio
Biotin-agagatccctcagacccttttagt
Linker
gtaatacgactcactatagggc
LAM-SB-L-20
acttaagtgtatgtaaacttccgact
LAM-LV-34
agacccttttagtcagtgtggaaa
Nested
agggctccgcttaagggac
LAM-LV-BC-OVH
acactctttccctacacgacgctcttccgatctAAAAagtcagtgtggaaaatctctagca
LAM-SBIII-BC-OVH
acactctttccctacacgacgctcttccgatctCCCAgtaaacttccgacttcaactgta
Illumina-1
Aatgatacggcgaccaccgagatctacactctttccctacacgacgctcttccgatct +
Illumina-2
caagcagaagacggcatacgagctcttccgatct+
Sonic TA Link(+)
gtaatacgactcactatagggctccgcttaagggaccatacgagctcttccgatct
Sonic Link(-)amino
gatcggaagagctcgtatga-Amino
+
Oligonucleotide sequences © 2006-2010 Illumina, Inc All rights reserved.
References
1.
Schmidt M, et al. (2007). High-resolution insertion-site analysis by linear amplification-mediated
PCR (LAM-PCR). Nat Methods 4: 1051-1057.