GSM8657479: Prime-seq splenic T cells; Mus musculus; RNA-Seq
RNA was harvested using an Rneasy mini kit (Qiagen). Twenty-five ng of RNA was used for cDNA synthesis. Dnase I digestion was performed in well, rather than on column. RNA libraries were constructed using  Prime-seq protocol (doi: 10.1186/s13059-022-02660-8). Briefly, extracted RNA was treated with DNAse I and then reverse transcribed into full-length cDNA using barcoded oligodT primers and template switching oligos. Following first strand synthesis, the samples were pooled, cleaned with magnetic beads, and the remaining primers were digested using Exonuclease I. Second strand synthesis and pre-amplification was performed, and the amplified cDNA was quantified using PicoGreen and qualified using a HS DNA chip on the Bioanalyzer (Agilent). High quality samples were used for library construction using the NEB Next Ultra II FS kit (NEB) according to prime-seq specifications. The libraries were quantified and qualified using the HS DNA chip on the Bioanalyzer (Agilent).