GSM1537117: single cell whole genome sequencing data from SKBR3 cell 3 obtained with the DR-seq protocol; Homo sapiens; OTHER
DR-Seq. First strand cDNA synthesis was performed by adding 2 μL of reaction mix containing 0.2 μL first strand buffer (MessageAmp II, Life technologies), 0.4 μL of dNTP mix (MessageAmp II, Life technologies), 0.1 μL Arrayscript (MessageAmp II, Life technologies), 0.1 μL RNAse inhibitor (MessageAmp II, Life technologies), 0.2 μL RT primer with cell specific barcode (Ad-1x)13, 0.2 μL 1:500000 diluted ERCC spike-in mix 1 (Life technologies) and 0.05% IGEPAL in water. The first strand cDNA synthesis and lysis reaction mix together with the spike-in molecules were added directly to the drop in the lid of the tube containing a single cell. Samples were incubated in a PCR machine with lid and block set to 42C for 15 minutes after which the samples were spun down and incubated for another 105 minutes. After first strand synthesis, samples were incubated for 10 minutes at 80oC. Quasilinear amplification buffer containing 6.0 μL ThermoPol buffer (NEB) 1.0 μL 10 mM dNTP mix, 26 μL water and 0.15 μL 50 μM primer mix (Ad-2)7 was added to each sample. Samples were incubated for 3 minutes at 94 oC to denature the DNA. Seven cycles of quasilinear amplification was performed (10oC for 45 seconds,15oC for 45 seconds, 20oC for 45 seconds, 30oC for 45 seconds, 40oC for 45 seconds, 50oC for 45 seconds, 65oC for 2 minutes, 95oC for 20 seconds, 58oC for 40 seconds and then immediately quench on ice). Prior to each cycle 0.6 μL polymerase mix containing 2U Bst large fragment (NEB) and 0.8U Pyrophage 3173 exo- (Lucigen) was added. Note that the 58oC step for 40 seconds step, prior to quenching the reaction on ice, is not performed for the first quasilinear amplification round. After 7 sounds of quasilinear amplification, samples were split in two. One half of the sample was processed for gDNA sequencing, the other half was processed for mRNA sequencing. For mRNA sequencing, second strand synthesis of the quasilinear amplified cDNA was performed using the P1 primer (5' - CGATTGAGGCCGGTAATAC - 3') in a single cycle of PCR (94oC for 20 sec, 51oC for 20 sec, 72oC for 7 min). After this, samples with non-overlapping barcodes were pooled and cleaned up on a cDNA purification column (MessageAmp II, Life technologies), and eluted twice with 9 μL of  water at 55oC. Next, the volume of the sample was reduced to 6.4 μL using a SpeedVac®. In vitro transcription (IVT) mix containing 1.6 μL 10x IVT buffer, 1.6 μL ATP, 1.6 μL GTP, 1.6 μL CTP, 1.6 μL UTP and 1.6 μL enzyme mix (MessageAmp II, Life technologies) were added to the samples and incubated at 37oC for 13 hours. After IVT, samples were cleaned up using the aRNA clean up columns (MessageAmp II, Life technologies) and aRNA was eluted twice in 12 μL of warm water at 55oC. After clean up, aRNA quality was assessed on a bioanalyzer (Agilent) Eukaryote Total RNA Pico chip. Library preparation was performed as previously described in Hashimshony et al. Cell reports 2012. For DNA sequencing, the other half of the quasilinear amplification product was amplified further by PCR. PCR mix containing 1.0 μL 10mM dNTP, 3 μL Thermopol buffer (10X), 0.2 μL 100 μM primer P2 (5' - GTGAGTGATGGTTGAGGTAGTGTGGAG - 3') and 1.0 μL Deep VentR (exo-) polymerase (NEB) was added to each sample for a final volume of 68 μL. PCR was performed as follows, 21 cycles of (94oC for 20 seconds, 59oC for 20 seconds, 65oC for 1 minute, 72oC for 2 minutes), and 72oC for 5 minutes at the end. After PCR, the quality of the products were assessed by agarose gel electrophoresis and the samples were cleaned up using a PCR purification column. Next, to remove adapter Ad-2 from the PCR product prior to preparing Illumina libraries, another PCR was done starting with 80 ng of product from the previous step. PCR mix containing 0.3 μL of 50 μM primer P3 with a 5' biotinylated end (5' - GTGAGCTGGAGTTGAGGTAGTGTGGAG - 3'), 5 μL Thermopol buffer (10X), 1 μL 10mM dNTP and 1 μL Deep VentR (exo-) polymerase (NEB) was added to each sample for a final volume of 50 μL. PCR was performed as follows, 94oC for 2 minutes, then 4 cycles of (94oC for 20 seconds, 46oC for 20 seconds, 65oC for 1 minute and 72oC for 2 minutes) and 9 cycles of (94oC for 20 seconds, 59oC for 20 seconds, 65oC for 1 minute and 72oC for 2 minutes). The PCR product was sheared using a sonicator (Biorupter®) on the low power setting with 15  cycles of 1 minute (30 seconds On, 30 seconds Off) with constant cooling at 4oC. The sheared products was then cleaned up using a PCR purification column (Qiagen) and eluted in 50 μL water. The final product distribution was verified on a bioanalyzer (Agilent) High Sensitivity DNA chip to have an average product size of approximately 300 bp. The DNA products was then added to Dynabeads MyOne Streptavidin C1 beads (Life Technologies) in 50 μL 2X BW buffer (10 mM Tris-HCl, 1mM EDTA and 2mM NaCl). After immobilizing the DNA products on the beads for 15 minutes, the biotinylated DNA was separated using a magnetic stand and the supernatant was stored. The biotinylated DNA was digested on the magnetic beads and the beads were washed twice with 50 μL  1X BW buffer. These two washes were then combined with the first supernatant and purified using a PCR purification column (Qiagen). Finally, Illumina libraries were prepared with different index primers for each single cell using the NEBNext Ultra DNA Library Prep Kit for Illumina® (NEB). Trypsinized single cells were picked using a mouth pipet with a 30μm glass capillary under a stereomicroscope. Picked cells were deposited in the center of the lid of a 0.2ml PCR tube and snap frozen in liquid nitrogen.
GEO Accession
GSM1537117