SETDB1 knock-out and reexpression of a WT, a catalytic-dead variant (CA) or a NLS-tagged SETDB1 protein

In order to generate the Setdb1 cKO allele, exons 15 and 16, which encode the core amino acids of the catalytic domain, have been flanked with two lox-P sites recognized by Cre recombinase enzyme. Cre-oestrogen receptor fusion gene Mer-Cre-Mer has been introduced to induce acute Setdb1 KO after Tamoxifen treatment. Setdb1 cKO mESCs where Setdb1 expression is stably rescued by wild type 3xFlag-Setdb1 (WT) or by the catalytic dead mutant 3xFlag-Setdb1 (CA) were generously given by Pr Yoichi Shinkai. The catalytic dead mutant was obtained with a single lysine mutation. In the lab, Setdb1 cKO mESCs, where Setdb1 expression is stably rescued by NLS-3xFlag-Setdb1 which localizes only in the nucleus, have been established (3 NLS sequences have been added in order to retain Setdb1 in the nucleus).

The quality of the RNA was determined on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), the RNA integrity number was above 8 for all the samples. To construct libraries, 1 mg of high-quality total RNA sample was processed using Truseq® stranded total RNA kit (Illumina®). After the removal of ribosomal RNAs (using Ribo-zero® rRNA), confirmed by QC control on pico chipTM on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), total RNA molecules are fragmented and reverse-transcribed using random primers. Replacement of dTTP by dUTP during the second strand synthesis will permit to achieve the strand specificity. Addition of a single A base to the cDNA is followed by ligation of adapters. Libraries were quantified by qPCR using the KAPA Library Quantification Kit for Illumina Libraries (KapaBiosystems) and library profiles were assessed using the DNA High SensitivityTMHS kit on an Agilent Bioanalyzer 2100. Libraries were sequenced on an Illumina® Nextseq 500 instrument using 75 base-lengths read V2 chemistry in a paired-end mode.