title: GSM5384945: 14Rik #1 total-RNA; Mus musculus; OTHER
library_construction_protocol: For total-RNA, >5M library-containing cells was pelleted and resuspended in Trizol (Ambion). RNA was cleaned up using Direct-zol RNA miniprep columns (Zymo Research), treated with RQ1 DNase (Promega), then cleaned up again using a total RNA purification kit (Norgen Biotek Corp). First, RNA was reverse transcribed with Superscript IV (Thermo Fisher), using a gene-specific primer that annealed 287nt downstream of the variable regions. cDNA was RNase-treated and then concentrated using 2x RNA XP beads (Beckman Coulter) to limit the number of parallel PCR reactions that had to be set up. Sequencing libraries were generated through 17-20 PCR cycles using NEBNext Ultra II Q5 (New England Biolabs) and NEBNext Multiplex oligos, uniquely indexing each sample. Amplicons were cleaned up using 1.5x AMPure XP beads (Beckman Coulter) and concentrations were quantified using the NEBNext Library Quant Kit for Illumina, before mixing indexed samples and checking the size distribution by TapeStation (Agilent).