GSM8781109: Control, Spleen, old, rep16; Mus musculus; RNA-Seq
RNA was isolated from TCL lysate via AmpureXP beads (Beckman Coulter). Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously (Fischer et al. 2023). Barcoded cDNA of each sample was generated with a Maxima RT polymerase (Thermo Fisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs) and an adaptor. 5'-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. cDNA from all samples was pooled and amplified with KAPA HiFi ReadyMix (KAPA Biosystems). To obtain sequencing libraries, tagmented and 3' ends were amplified with the Nextera XT Kit (Illumina). The library was sequenced on a NextSeq 500 (Illumina) with 16 cycles for the barcodes and UMIs in read1 and 51 cycles for the cDNA 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 (GRCh38) was used for alignment. Transcript and gene definitions were used according to GENCODE M25.