GSM3048121: Cell_AAGCCACGCATACGA; Homo sapiens; RNA-Seq
Differentiating hCA3 population were dissociated into a single cell suspension by enzymatic dissociation by treating with Accutase (Stem cell technologies). Cells were collected in PBS containing 0.1%BSA on ice. The cell suspension obtained was filtered with 22µm filter (Millipore) and kept in cold PBS-BSA solution. Before counting, trypan blue was added to the cell suspension and the viability, cell density and presence of clumps were assayed on a TC20™ Automated Cell Counter. Cell suspension at a final dilution of 2500 cells/µL and containing no less than 85% live cells were taken for the encapsulation and later steps. All reagents for encapsulation, cDNA synthesis, library preparation and sequencing were from Illumina inc. Cell suspensions were loaded into a ddSEQ catridge in a ratio of 21.5 µL Cell enzyme mix to every 4.5 µL cell suspension. 60ul of 3' barcode suspension mix and encapsulation oil were also loaded on the ddSEQ cartridge. The chip was then processed on a ddSEQ™ Single-Cell Isolator for about 5 minutes to generate single cell droplets. Encapsulated samples were transferred to a 96 well plate and reverse transcription mix was added and followed by reverse transcription on a thermal cycler. When the reverse transcription was done droplet disruptor was added to the product. This was followed by the first strand purification using magnetic beads. The beads were immobilized using magnetic peg stand and dynamag and supernatant was discarded. The beads were washed twice with 80% EtOH. 30uls of resuspension buffer was added for elution and supernatant was transferred to a new tube. The purified first strand was used to synthesize the second strand to generate the double strand cDNA. Again the cDNA was purified using magnetic beads, followed by 80% EtOH wash and elution. Amplified cDNA was simultaneously fragmented and barcoded by tagmentation using Tagment enzyme on thermal cycler. DNA adapters were added for cluster formation. Followed by the index addition, libraries were cleaned up using magnetic beads as before. Purified libraries were eluted with 20ul resuspension buffer. 1ul of eluted product was assessed on an Agilent BioAnalyzer. Library size was about 500bp. Sequencing was performed on an Illumina HiSeq 2500 instrument using surecell-sequencing primers.
GEO Accession
GSM3048121