BAZ_ChIP
For ChIP assays, chromatin immunoprecipitation was performed according to the method described previously8 with some modifications. Briefly, young adult or aged worms were harvested and washed several times in M9 buffer, and then washed twice in PBS buffer. Worms were lysed in cross linking buffer (1% formaldehyde in PBS with proteinase inhibitor cocktail) using a glass dounce homogenizer on ice, and fixed on a Nutator shaker for 15 min at 37C. Fixed worm samples were quenched by 0.125 M glycine and washed with cold PBS buffer with protease inhibitors for three times. Then samples were re-suspended in ice-cold FA buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail) with 0.1% SDS, and sonicated using Bioruptor sonication system (Diagenode UCD-200) at high amplitude for 10-15 cycles of 30 s on and 30 s off. The sonicated samples were centrifuged at 16000 g for 15 min at 4 C. Then, samples were pre-cleaned with protein A/G agarose beads and immunoprecipitated overnight using GFP-trap agarose beads (Chromotec ACT-CM-GFA0250) at 4 C. After proteinase K digestion and reverse cross-linking, the precipitated DNA and input DNA were purified using phenol-chloroform, precipitated with ethanol and subjected to DNA library construction or ChIP-qPCR assays. For ChIP-seq analysis of histone H3K9 methylation, digestion of chromatin was performed using 6 l of Micrococcal nuclease (MNase) (CST #10011S) in 400 l of Buffer B (CST #7007)containing 0.5 mM DTT for 20 min at 37 C, and then was stopped by 25 mM EDTA. About 10g of the digested chromatin was used for immunoprecipitation, and 5% of the extract was saved as input sample. The antibodies used were H3K9me1 (Abcam ab9045), H3K9me2 (Abcam ab1220) and H3K9me3 (Abcam ab8898).For ChIP-seq library preparation, 20 ng purified DNA samples were used. Library construction was performed using the QIAseq Ultralow Input Library Kite (QIAGEN 180495) following the manufacturer recommendations. DNA libraries were sequenced on Illumina HiSeq X-ten instrument, with 150-bp pair-end sequencing. For ChIP-qPCR analysis of genes bound to BAZ2B or EHMT1 in HEK293T cells. Immunoprecipitation was performed using anti-IgG, anti-FLAG (Sigma, F3165) or anti-EHMT1 (R&D SYSTEMS, PP-B042200) antibodies. Immunoprecipitated DNA was purified using phenol/chloroform extraction and then used for qPCR detection.