GSM5290371: non-CD41+CD42+_cells; Homo sapiens; RNA-Seq
The procedure of scRNA-seq followed the C version 3.1 from Steve McCarroll's lab (http://mccarrolllab.org/wp-content/uploads/2015/05/Online-Dropseq-Protocol-v.- 3.1-Dec-2015.pdf). Briefly, 1 mL of the samples containing approximately 100,000 cells with 90% viability were processed using microfluidic devices (PMDS Microfluidic Devices, FlowJEM). 8,000~10,000 cells were encapsulated in microfluidic droplets with 3000~4000 single cells recovered after washing and enzymatic steps. The resultant emulsion droplets were broken and subject to reverse transcription, followed by 14 cycles of PCR amplification---95 °C for 3 min, 4 x (98 °C for 20 s, 65 °C for 45 s, 72 °C for 3 min), 10 x (98 °C for 20 s, 67 °C for 20 s, 72 °C for 3 min), and 72 °C for 5 min. After purification with AMPure XP beads (Beckman Coulter), 600 pg barcoded cDNA was fragmented by Amplicon Tagment enzyme using Nextera XT Kit (Illumina), followed by indexing PCR--95 °C for 30 s, 12 x (95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s), and 72 °C for 5 min. The resulting DNA library was further purified with 0.6 x AMPure XP beads before sequencing. Libraries were sequenced on Illumina NextSeq 500 platform (R1- 20 cycles, index1- 8 cycles, R2- 64 cycles). Samples were demultiplexed and raw fastq data were generated using bcl2fastq2 v2.19