GSM8795642: primary human hepatocyte, 025, rep1; Homo sapiens; RNA-Seq
Cell suspensions (300-600 living cells per microliter determined by Count Star) were loaded on a Chromium Single Cell Controller (10× Genomics) to generate single-cell gel beads in emulsion (GEMs) by using Single Cell 3 Library and Gel Bead Kit V2 (10× Genomics, 120237) and Chromium Single Cell A Chip Kit (10× Genomics, 120236) according to the manufacturer's protocol. Briefly, single cells were suspended in 0.04 % BSA-PBS. About 8,700 cells were added to each channel with a targeted cell recovery estimate of 5,000 cells. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. GEMs were reverse transcribed in a C1000 Touch Thermal Cycler (Bio Rad) programed at 53 °C for 45 min, 85 °C for 5 min, and hold at 4 °C. After reverse transcription, single-cell droplets were broken and the single-strand cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNA was generated and amplified, and quality was assessed using the Agilent 4200. After the expansion of the material passes quality inspection, the construction of a sequencing library is carried out. First, cDNA is fragmented into fragments of about 200-300 base pairs (bp) using chemical methods. The cDNA fragments are then subjected to end repair and adenylation, followed by cDNA fragment selection. P7 Adapters are ligated to connect the fragments, and PCR amplification is used to introduce sample indices. Finally, fragment selection is performed to obtain the cDNA library. After the library is completed and passes quality inspection, sequencing is carried out using the Illumina NovaSeq sequencing platform to obtain sequencing data, which is then followed by subsequent data analysis. Illumina basecall files (*.bcl) were converted to fastqs using the Cell Ranger pipeline with recommended parameters.