Total RNA-seq of DNMT3a/3b induced double knockouts in mouse embryonic fibroblasts
RNA extraction was performed with the Qiagen AllPrep DNA/RNA Mini Kit (cat no. 80204). Libraries were generated using the NEBNext rRNA Depletion Kit (NEB, cat no. E6310) and Ultra II Directional RNA Library Prep Kit for Illumina (NEB, cat no. E7760) with the following specifications. RNA Integrity Number (RIN) was determined using the Agilent RNA 6000 Pico Kit (cat no. 5067-1513) on the Agilent 2100 Bioanalyzer and its associated software. 1g of RNA was hybridised to probes for rRNA depletion, treated with RNase H and DNase I, and purified before being fragmented at 94C with incubation times ranging from 8 to 15 minutes depending on the RIN. The fragmented RNA was then reverse-transcribed to cDNA in two steps and purified. Following end repair and adapter ligation, the cDNA libraries were purified, PCR enriched for 9 cycles, and purified again. All purification steps took place using Ampure XP beads (Beckman Coulter, cat no. A63881). The final libraries were quality checked and quantified for multiplexing using the Bioanalyzer High-Sensitivity DNA kit (Agilent, 5067-4626), Qubit dsDNA HS Assay kit (Thermo Scientific, Q32854), and the KAPA Library Quantification Kit optimised for Roche LightCycler 480 (Roche, 07960298001). The multiplexed libraries were sequenced as 150 bp paired-end reads on the Illumina Novaseq 6000 S1.