GSM8554025: Mouse LZ GFP-cMYCpos short-read rep5; Mus musculus; RNA-Seq
For germinal centre B cell populations, cell lysates from were kept in 22.5 μl RLT plus buffer at -80 °C. These were thawed on ice, and where necessary to provide sufficient cells, pooled. The RNA was then purified using a ratio of 0.6x Agencourt RNAClean XP (Beckman Coulter- A63987); RNA was eluted in 4 μl nuclease-free H2O. 0.5 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. The remaining RNA was split equally between short- and long-read library generation. 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