GSM5620896: Bulk_6h_N2-33034; Homo sapiens; RNA-Seq
For scRNA-seq, the 10x Genomics ChromiumTM controller and Single Cell 3' Reagent Kits v2 (Pleasanton, CA, USA) were used to generate single cell libraries. Briefly, cells were counted following FACS and cell suspensions were loaded for a targeted cell recovery of 1000-5000 cells per channel.The microfluidics platform was used to barcode single cells using Gel Bead-In-Emulsions (GEMs). For bulk RNA-seq, RNA was extracted using Trizol (Thermo). For scRNA-seq, RT is performed within GEMs, resulting in barcoded cDNA from single cells. The full length, barcoded cDNA is PCR amplified followed by enzymatic fragmentation and SPRI double sided size selection for optimal cDNA size. End repair, A-tailing, Adaptor Ligation, and PCR are performed to generate the final libraries that have P5 and P7 primers compatible with Illumina sequencing. The libraries were pooled and sequenced using an Illumina NextSeq500 platform with a 150 cycle High Output v2 kit in paired-end format with 26 bp Read 1, 8 bp I5 Index, and 85 bp Read 2. For bulk RNA-seq, SMARTer Stranded Total RNA Sample Prep Kit - HI Mammalian (Takara) was used to generate cDNA libraries for sequencing. The libraries were pooled and sequenced using an Illumina NextSeq500 platform with a 150 cycle High Output v2 kit in single-end format with Read 1: 85 cycle, Read 2: 0 cycle, and single index: 8 cycle. Following sequencing, FASTQ files were generated from the raw sequencing data using the BaseSpace Onsite (Illumina).