GSM8958826: KP primary cell line with Rnf2 knockdown, shRnf2-3, RNF2 CUT&RUN; Mus musculus; OTHER
CUT&RUN was performed using the Epicypher CUTANATM ChIC/CUT&RUN Kit (14-1048) according to the manufacturer's specifications with the following conditions and modifications: For binding to the Concanavalin beads, 500,000 cells per sample were prepared, counted twice by hemacytometer, and averaged. Nuclei were prepared according to the Epicypher Cut and Run Manual Appendix. Nuclei were incubated with activated beads. 0.1 E. coli spike in DNA ng was added to each sample. The following antibodies (0.5 ug of antibody per reaction) were used:IgG Control antibody: CUTANATM Kit Rabbit IgG CUT&RUN Negative Control Antibody; RNF2 antibody: CST RING1B (D22F2) XP® Rabbit mAb #5694; H3K4me3 antibody: Epicypher Rabbit Polyclonal H3K4me3 13-0041; H3K27me3 antibody: CST Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733; H2AK119ub antibody: CST Ubiquityl-Histone H2A (Lys119) (D27C4) Rabbit mAb #8240; BMI-1 antibody: Active Motif BMI-1 antibody (mAb) 39993; RNA Pol II p-ser5: Abcam Anti-RNA polymerase II phosphor-S5 EPR19015 Libraries were prepared using the Epicypher CUTANATM CUT&RUN Library Prep Kit (14-1001) according to the manufacturer's specifications with the following modification: SPRI-select beads were used after library preparation to perform 200-700 bp size selection to enrich for DNA fragments from CUT&RUN and remove any adapter dimers or high molecular weight DNA. Library quality was analyzed using Agilent Tapestation D1000 High Sensitivity Tapes (#5067-5584). Sequencing was performed with 10 million reads ordered per sample, 150 bp paired-end reads, on an Illumina NovaSeq 6000.