GSM3602455: AI_H9 [WAM464A6_S6]; Homo sapiens; RNA-Seq
cDNA was generated from single cells using the SMART-Seq V4 Ultra Low Input Kit (Clontech 634898) as previously described (Blakely et al. 2015). cDNA was amplified by adding 25 μl 2X SeqAmp PCR buffer, 1 μl PCR Primer II A (12 μM), 1 μl SeqAmp DNA polymerase, 3 μl nuclease-free water, and incubated on a PCR machine at 95°C for 1 min, 23 cycles of 98°C for 10 s, 65°C for 30 s and 68°C for 3 min, before a final extension for 10 min at 72°C. Amplified cDNA was purified using 50 μl AMPure XP beads (Beckman Coulter) per manufacturer's instructions. Sample tubes were mixed well and incubated at RT for 8 min to bind the DNA to the beads. Tubes were put on a magnet until the supernatant ran clear so that it could be removed and discarded. The beads were washed twice with 200 μl 80% ethanol. Any remaining ethanol was removed and beads allowed to dry, then resuspended in 12 μl of elution buffer. Tubes were returned to the magnet and the clear supernatant containing the cDNA was collected in a new tube and stored at −80°C until library preparation. cDNA quality was assessed by High Sensitivity DNA assay on an Agilent 2100 Bioanalyser, with good quality cDNA showing a broad peak from 300 to 9000 bp. cDNA concentration was measured using Qubit dsDNA HS kit (Life Technologies). In preparation for library generation, 10 μl of cDNA sample and 32 μl purification buffer were added to a Covaris microTUBE AFA Fiber Pre-Slit Snap Cap. cDNA was sheared to a 200-500 bp range using an E220 focused-ultrasonicator (Covaris) for 2 mins at Peak Incident power 175 W, Duty Factor 10%, 200 cycles per burst, water level 5. Libraries were prepared using a Low Input Library Prep Kit HT (Clontech, 634900) per manufacturer's instructions. Library quality and DNA concentration was assessed by Bioanalyser and Qubit. For library purification, 75 μl of AMPure XP beads was added to each collection tube containing the genomic DNA and treated as described above.
GEO Accession
GSM3602455