GSM4475599: PFC_CTR_05; Mus musculus; RNA-Seq
RNA sequencing libraries were prepared using 50 ng of total RNA samples following BRB-seq protocol (Alpern et al., 2019). Briefly, each RNA sample was reverse transcribed in a 96-well plate using SuperScriptTM II Reverse Transcriptase_x000B_with individual barcoded oligo-dT primers. Next, all the samples were pooled together, column purified and treated with exonuclease I. Double-stranded cDNA was generated by the second stand synthesis with DNA polymerase I following the nick translation method. Full-length double-stranded cDNA was purified with AMPure XP magnetic beads and eluted in 20 μL of water. The Illumina compatible libraries were prepared by tagmentation of 5 ng of full-length double-stranded cDNA with in-house produced Tn5 enzyme. The final library was amplified 15 cycles and the fragments ranging 200–1000 bp were size-selected using AMPure beads. The libraries were sequenced using the Illumina NextSeq 500 platform with a custom primer and the High Output v2 kit. The library loading concentration was 2.2 pM and sequencing configuration as following: R1 6c / index 8 c / R2 70c.