GSM7592893: S3-CTRL_HRO_3; Homo sapiens; RNA-Seq
21.8 µl of the cells suspension (an estimated number of 9,000-11,000 cells) were carefully mixed with reverse transcription reaction mix using the Chromium Single Cell 3' Library & Gel beads chemistry v2 and loaded into a Chromium Single Cell A Chip (10X Genomics, Pleasanton, CA). During the encapsulation process in the 10X Genomics Chromium system (Zheng et al., 2017), the cells lysed within the droplet and released polyadenylated RNA bound to the barcoded bead that was captured with the cell. Following the guidelines of the 10X Genomics user manual, the droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using Dynabeads MyOne Silane (ThermoFisher Scientific). After the amplification of cDNA with 10 cycles, it underwent purification and a quality control check on the Fragment Analyzer. The cDNA was fragmented for five minutes and dA-tailed, followed by an adapter ligation step and an indexing PCR of 10 cycles in order to generate libraries. After quantification, the libraries were sequenced on an Illumina NextSeq 500 machine using a high-output flowcell in PE mode (R1: 26 cycles; I1: 8 cycles; R2: 57 cycles), thus generating 220 mio. fragments at minimum per sample.