GSM5219148: 1m spleen (m-T cells); Mus musculus; RNA-Seq
Cells were sorted on a BD Aria III into TCL buffer and frozen at -80°C. Total RNA was isolated using the RNAeasy Plus micro kit (Qiagen, #74034). Quality and integrity of total RNA was controlled on a Bioanalyzer 2100 (Agilent Technologies). Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously (Parekh et al., 2016). Briefly, barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher, #EP0742) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. Ends of the cDNAs were extended by a template switch oligo (TSO) and full-length cDNA was amplified with primers binding to the TSO-site and the adaptor. NEB UltraII FS kit was used to fragment cDNA. After end repair and A-tailing a TruSeq adapter was ligated and 3'-end-fragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al. (2016), the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 67 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2. Data was processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables (Macosko et al., 2015). Reference genome (GRCm38) was used for alignment. Transcript and gene definitions were used according to GENCODE version M25