GSM8554010: Mouse naïve short-read rep5; Mus musculus; RNA-Seq
1x10^5 naive B cells isolated by FACS were directly sorted into an Eppendorf tube containing 350 μl RLT plus buffer (Qiagen-1053393). Cells were homogenized with gentle pipetting and 1 volume ratio of 80% ethanol was added. The rest of RNA isolation was conducted according to the protocol of RNeasy Plus Micro Kit (Qiagen-74034). RNA was eluted in 11 μl nuclease-free H2O. 1 μl was used to assess RNA quality using the Agilent 2100 Bioanalyzer, with RIN > 7.5 used as a threshold for inclusion in library generation. 1.5 μl RNA was then used for short-read library generation, with a further 1.5 μl used to generate corresponding long-read libraries. See Samples section cDNA was generated using the SMARTseq v4 ultra low input RNA kit for sequencing; 0.03 pg SIRV/ERCC spike-in RNA (https://www.lexogen.com/store/sirv-set4/) was added into the reverse transcription reaction for each sample. Libraries were then prepared from the cDNA using the Nextera XT DNA library preparation kit. Note that for three samples (Mouse LZ GFP-cMYCneg rep3; LZ GFP-cMYCpos rep1 and LZ GFP-cMYCpos rep2), initial library preparation failed. Nuclease-free water was therefore added to the tubes in order to retrieve all remaining RNA, and library preparation repeated, resulting in measurable libraries