GSM3891428: PRC1CKO_UNT_RNA_rep3; Mus musculus; RNA-Seq
For cRNA-seq, 1 x 10^7 mouse ESCs (both untreated and following 72 hours OHT treatment) were mixed with 4 x 10^6 Drosophila SG4 cells in 600 ml PBS. For RNA extraction, 400 ml of cells was used (corresponding to 6.7 x 10^6 mouse ESCs), and for DNA extraction the remaining 200 ml of cells was used (corresponding to 3.3 x 10^6 mouse ESCs). RNA extraction was performed using RNeasy mini kit columns (Qiagen) following manufacturer's guidelines, and 10 mg was subjected to Turbo DNase (ThermoFisher) treatment to remove any contaminating DNA. Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent). Next, RNA samples were depleted of rRNA using the NEBNext rRNA Depletion kit (NEB). To quantitate the consistency of spike-in cell mixing for each individual sample, a matched sample of cells was used to isolate genomic DNA using Quick-DNA miniprep kit (Zymo). RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep kit (NEB). Libraries from gDNA were prepared using NEBNext Ultra II FS kit (NEB) following manufacturer's guidelines. RNA and DNA libraries were sequenced as 80 bp paired-end reads on the Illumina NextSeq 500 platform.