title: GSM8682965: gDNA siNT 3; Mus musculus; OTHER
library_construction_protocol: For gDNA-derived samples: Genomic DNA was extracted using QuickExtract (Lucigen). For total-RNA-derived samples: Cells was pelleted and resuspended in Trizol (Ambion), spike-in RNA was added. 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). For gDNA-derived samples: In a first round of PCR, the Oct4 uaRNA locus was amplified from these samples with NEBNext Ultra II Q5, using primers that would not amplify remaining plasmid or aspecific integrations at other loci. Amplicons were cleaned up using 0.9X AMPure XP beads. The amplicons from the first round of PCR were used as template in a PCR with the NEBNext multiplex oligos to generate the sequencing library. Final amplicons were cleaned up using 1x AMPure XP beads (Beckman Coulter), and concentrations were quantified using the NEBNext Library Quant Kit for Illumina, before mixing indexed gDNA samples together. After pooling the sample was gel-extracted from a 1% agarose gel to size select. For RNA-derived samples: First, RNA was reverse transcribed with Superscript IV (Thermo Fisher), using a gene-specific primer in mKate2 annealed 221nt 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 PCR 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).