title: GSM8377239: RNA-Seq CD4_Memory_Y_5; Mus musculus; RNA-Seq
library_construction_protocol: RNA-seq Poly-A based RNA sequencing libraries were generated as described by us (Sun et al.  Nat Comms 2021;  https://doi.org/10.1038/s41467-021-22863-0.). In brief, a 3-prime biased approach was used to generate sequencing libraries. An 8 bp sample index and a 10 bp unique molecular identifier (UMI) were added during initial poly(A) priming and pooled samples were amplified using a template-switching oligonucleotide. The Illumina P5 (5′-AAT GAT ACG GCG ACC ACC GA-3′) and P7 (5′-CAA GCA GAA GAC GGC ATA CGA GAT-3′) sequences were added by PCR and Nextera transposase, respectively. The library was designed so that the forward read (R1) utilises a custom primer (5′-GCC TGT CCG CGG AAG CAG TGG TAT CAA CGC AGA GTA C-3′) to sequence directly into the index and then the 10 bp UMI. The reverse read (R2) uses the standard R2 primer to sequence the cDNA in the sense direction for transcript identification. For RNA sequencing, a 3-prime biased approach was used to generate sequencing libraries as described previously (Sun et al.  Nat Comms 2021;  https://doi.org/10.1038/s41467-021-22863-0.). Barcoded 19bp R1 and 111bp R2 paired-end reads per sequencing lane were assigned to individual samples using the sabre software (version 80faf94) with options “-u –m2 –l 10' (https://github.com/najoshi/sabre) (Tsyganov et al. JOSS 2018; https://doi.org/10.21105/joss.00583).