Shirley Zhu/ cDNA library construction 3/10/2014
-In a 0.2 ml PCR tube, mix: mRNA (about 1ng-50ng)
Primer RT_P7dT_Index (10uM)
Total volume
Index Bar code (see Page6 for the sequence information):
9ul
1ul
10ul
Index_2
Index_4
Index_6
Index_8
(For Hi-Seq machine, 1 lane includes 4 samples with index bar codes.)
-Mix well; incubate the tube at 85C for 5 min, then cool down to 50C in thermal cyclers.
-In a 0.2 ml PCR tube, mix: mRNA (about 1ng-50ng) 9ul
Primer P7_oligodT (10uM) [We order from IDT]
First strand buffer (Invitrogen CAT#18080-044)
Total volume
1ul
4ul
14ul
-Mix well; For high quality RNA (ex. from frozen tissue, cell line), incubate the tube at
85C for 5-10 min, then cool down to 50C in thermal cyclers. For degraded RNA from
FFPE, incubate the tube at 85C for 3-5 min, then cool down to 50C in thermal cyclers.
-to each 10ul of mRNA, add: (keep the samples at 50C)
[Don’t add first strand buffer if previously added during heat shearing]
First strand buffer (Invitrogen CAT#18080-044)
0.1M DTT
10mM total dNTP (2.5mM each dNTP)
RNaseOUT (Invitrogen CAT#10777-019)
Superscript III Reverse Transcriptase
(Invitrogen CAT#18080-044)
4ul
2ul
1ul
1ul
2ul
10ul
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Shirley Zhu/ cDNA library construction 3/10/2014
-make total volume to 20ul
-Incubate at 50C for 1 hour, then followed by 85C for 15 min to inactivate enzymes.
-to 20ul of 1 st
strand cDNA synthesis solution, add:
H2O
5X 2 nd strand buffer (Inv. CAT#10812-014)
10mM dNTP
E.coli DNA ligase(Inv. CAT#18052-019)
E.coli DNA polymerase I
(New England Biolab, CAT# M0209L)
E.coli RNaseH (Epicentre, Cat# R0601K)
make total volume to 150ul
-Incubate at 16C for 2 hours
91ul
30ul
3ul
1ul
4ul
1ul
130ul
-add 2ul of T4 DNA Polymerase (New England Biolab, CAT# M0203S)
-Incubate at 16C for 15 min
-add 10ul of 0.5M EDTA pH 8.0 to each tube to stop the 2 nd
strand synthesis reaction
-the volume now is 162ul
(SAFE STOPPING POINT: can store the samples at 4°C for overnight)
-add 486ul (3 volumes of 162ul the double stranded DNA) of buffer ERC or PB, mix well
-apply the sample to the columns
-Centrifuge at 14K rpm for 1 min, discard the flow-through
-add 750ul wash buffer PE to the column and centrifuge same as above, repeat once;
-dry the columns by 14K rpm for 3 min,
-transfer the column at a 1.5ml clean tube
-elute with 33.5ul of buffer EB, stand at RT for 1min
-Centrifuge at 14K rpm for 1 min
-Reload once, to get 32ul of dsDNA
(SAFE STOPPING POINT: can store the samples at 4°C for overnight or at -15° to -
25°C for up to seven days.)
-Prepare the following reaction mix:
Klenow buffer (NEB buffer 2, B7002S) 5ul
1mM dATP 10ul
Klenow exo (3’ to 5’ exo minus, M0212L)
3ul
18ul
-add to 32ul of DNA sample
-the total volume is 50ul
incubate the 50ul at 37 C for 30 min.
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Shirley Zhu/ cDNA library construction 3/10/2014
(Use 3 volume of PBI buffer)
-Add 150ul of PB or ERC buffer to 50ul of DNA samples
-apply the sample to the columns
-Centrifuge at 14K rpm for 1 min, discard the flow-through
-add 750ul wash buffer PE to the column and centrifuge same as above, repeat once;
-dry the columns by 14K rpm for 3 min,
-transfer the column at a 1.5ml clean tube
-elute with 11.5ul of buffer EB, stand at RT for 1min
-Centrifuge at 14K rpm for 1 min
-Reload once, to get 10ul of DNA fragment
(SAFE STOPPING POINT: can store the samples at 4°C for overnight or at -15° to -
25°C for up to seven days.)
-Prepare the following reaction mix on ice:
2XDNA ligase buffer (B2200S) 15ul
Adapter Oligo Mix
(From Illumina, Part# 1001782)
(1:50 dilution)
DNA ligase (M2200L)
-Add to 10ul of DNA
-The total volume is 30ul
-Incubate at 22C for 15 min
2ul
3ul
20ul
-Add 20ul of H2O to 30ul of the ligated DNA, total volume should be 50ul.
-Add an equal amount well mixed SPRI beads to the 50ul of ligated DNA. Mix well by pipette up and down for 10 times
-Sit for 5 min at room temperature.
-Place the tube in a magnetic plate and keep for 2 min., discard the supernatant.
-Add 300ul of 70% Ethanol to the tube without disturbing the beads, incubate for 1min at room temperature, and discard the supernatant. Repeat once.
-Air dry the beads for 5-10 min.; be sure to remove all of the ethanol.
-Off the magnetic rack.
-Add 30ul of EB buffer to the beads.
-Pipette mixing the beads for 10 times
-Sit at room temperature for 2 min.
-Place the tube to the magnetic rack and keep for 2 min.
-Transfer the 40ul of supernatant to a new 1.5ml tube. (LoBind tube, Eppendorf, Cat#
022431021)
(SAFE STOPPING POINT: store the samples at -15° to -25°C for up to seven days.)
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Shirley Zhu/ cDNA library construction 3/10/2014
-Prepare 100ul PCR reaction mix on ice:
2X phusion PCR master mix (NEB M-0531S)
PCR primer_F (from IDT),10uM/ul
PCR primer_R_index (from IDT) 10uM/ul
Total
50ul
5ul
5ul
60ul
PCR primer Index Bar codes have to match the Primer RT_P7dT_Index:
PCR_Index_2 to P7dT_Index_2
PCR_Index_4 to P7dT_Index_4
PCR_Index_6 to P7dT_Index_6
PCR_Index_8 to P7dT_Index_8
-Add to 40ul of adapter-ligated DNA,
-total volume is 100ul
(Always has a negative control with H2O for PCR reaction)
-Amplify with 15-cycle PCR with following conditions:
98C/30sec
15 cycles of (98C/10sec, 65C/30sec, 72C/30sec)
72C/5min
10C/hold
-Add an equal amount (100ul) well mixed SPRI beads to the 100ul of PCR products of cDNA libraries. Mix well by pipette up and down for 10 times
-Sit for 5 min at room temperature.
-Place the tube in a magnetic plate and keep for 2 min., discard the supernatant.
-Add 300ul of 70% Ethanol to the tube without disturbing the beads, incubate for 1min at room temperature, and discard the supernatant. Repeat once.
-Air dry the beads for 5-10 min. be sure to remove all of the ethanol.
-Off the magnetic rack.
-Add 30ul of EB buffer to the beads.
-Pipette mixing the beads for 10 times
-Sit at room temperature for 2 min.
-Place the tube to the magnetic rack and keep for 2 min.
-Transfer the supernatant to a new 1.5ml tube. (LoBind tube, Eppendorf, Cat# 022431021)
(SAFE STOPPING POINT: can store the samples at 4°C for overnight or at -15° to -
25°C for up to seven days.)
-Run the library PCR products on 3% Nusieve GTG agarose gel.
-Prepare 3% Nusieve GTG agarose gel:
-3g of GTG agarose in 100ml of 1X TBE buffer,
-stir 15min at room temperature
-Heat at a hot plate with stirring; cover the beaker.
-bring the solution to a boil while stirring
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Shirley Zhu/ cDNA library construction 3/10/2014
-Maintain gentle boiling until all the agarose are dissolved; about 10-15min
-cool the solution to 50-60C prior to casting
-50ml for a double tray, 25ml for a single tray
**Using Submarine-type electrophoresis system, Mupid-2plus (Helixx
Technologies, Inc)
-Casting gels for at least 40 min.
-Running condition: full voltage (100v), running time about 1.5 hour
-running buffer: 0.5X TBE, about 400ml for one unit.
-Excise the band in the 200-300 bp; using 25bp DNA ladder (Invitrogen, Cat# 10488-022)
-Purify the 200-300bp band using QIAquick Gel extraction kit, (Qiagen, CAT# 28704).
-Weigh the gel slice in a 15ml conical tube
-Add 6 volume buffer QG (eg. 600ul QG to 100mg gel)
-Allow the gel completely dissolve at room temperature, about 15-30min.
-Add one volume (eg. 100ul for 100mg gel) of Isopropanol to the sample and mix well by inverting the tube.
-Apply the sample (750ul) to the column and centrifuge at 14K rpm for 1 min, discard the flow-through.
-Repeat as many times as necessary until all solution loaded.
-Add 500ul QG to the column and centrifuge at 14k rpm for 1min to remove residual agarose. Discard the flow-through
-Add 750ul wash buffer PE to the column and centrifuge at 14K rpm for 1 min. Repeat once.
-Centrifuge the column at 14k for 3min. to remove residual wash buffer;
-Transfer the column to a clean 1.5ml tube, add 32ul buffer EB to the column to elute the
DNA, stand at RT for 1min.
-Centrifuge the column at 14K rpm for 1 min.
-Reload once; get 30ul of purified amplified adapter-ligated DNA
-Qubit (Qubit® 2.0 Fluorometer , Life Technologies) for OD measurement.
Each sample has to be exact amount:
Sample1 with Index_2 30-100ng
Sample2 with Index_4
Sample3 with Index_6
Sample4 with Index_8
30-100ng
30-100ng
30-100ng
(A minimum of 30 ng per sample)
-give a new name for the pooling sample.
-Qubit again to get the concentration.
-Ready for the deep Sequencing with Illumina HI-Seq
Index Bar code sequence for submission:
Index_2: CGATGT
Index_4: TGACCA
Index_6: GCCAAT
Index_8: ACTTGA
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Shirley Zhu/ cDNA library construction 3/10/2014
Oligonucleotides ordered from IDT:
RT Primers (RNase-free HPLC purify)
RT_P7dT_Index 2
5’
CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTG
CTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTTTTT*V*N 3’
RT_P7dT_Index 4
5’
CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTG
CTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTTTTT*V*N 3’
RT_P7dT_Index 6
5’
CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTG
CTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTTTTT*V*N 3’
RT_P7dT_Index 8
5’
CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTG
CTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTTTTT*V*N 3’
PCR Primers (HPLC purify)
PCR_F
5’
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCC
GATCT 3’
PCR_R _Index 2
5’ CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTC 3’
PCR_R _Index 4
5’ CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTC 3’
PCR_R _Index 6
5’ CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTC 3’
PCR_R _Index 8
5’ CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTC 3’
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