Illumina HiSeq 2000 sequencing; Epigenetics profiling of human BLaER1 pre-B cells transdifferentiation into macrophages
Epigenetics profiling of human BLaER1 pre-B cells transdifferentiation into macrophages
Cells were collected at twelve time points after transdifferentiation induction: 0, 3, 6, 9, 12, 18, 24, 36, 48, 72, 120 and 168 hours. Cell pellets were frozen prior to RNA extraction. Human BLaER1 cells were grown in RPMI medium (Invitrogen) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco), 2 mM L-glutamine, and 100 U/ml penicillin G sodium (Gibco). Cells were maintained at 37ºC in a humid atmosphere containing 5% CO2. Transdifferentiation was induced by addition of 100 nM of beta-estradiol (Calbiochem) and 10 nM of IL-3 and CSF-1 (Peprotech). For the ChIP-seq experiments of the nine histone marks and cEBPalpha, cells were crosslinked with formaldehyde 1 % (Sigma) for 10' at room temperature. The reaction was stopped by adding glycine to 0.25 M final concentration for 10' at room temperature. Fixed cells were resuspended in 100 ul of lysis buffer (SDS 1 %, EDTA 10 mM, TrisCl 50 mM and protease inhibitors). The lysate was sonicated for 25' using Covaris S2 system in TC12 tubes (Duty cycle 20\%, Intensity 8, cycles/burst 200, water level 15). The cleared supernatant was used immediately in ChIP experiments or stored at -80ºC. 5 ug of sonicated chromatin were diluted in 900 ul RIPA buffer - H3K4me3, H3K9ac, H4K20me1, H3K27me3 and H3K27ac (140 mM NaCl, 10 mM Tris HCl pH 8.0, 1 mM EDTA, 1 % Triton X-100, 0.1 % SDS, 0.1 % Na deoxycholate, protease inhibitors) -, RIPA 2X - H3K4me1, H3K4me2 and H3K9me3 (280 mM NaCl, 10 mM Tris HCl pH 8.0, 1 mM EDTA, 2 % Triton X-100, 0.2 % SDS, 0.2 % Na deoxycholate, protease inhibitors) -, or RIPA 1X 1 % triton - H3K36me3 (280 mM NaCl, 10 mM Tris HCl pH 8.0, 1 mM EDTA, 1 % Triton X-100, 0.2 % SDS, 0.2 % Na deoxycholate, protease inhibitors). For H3K4me3, H3K36me3, H3K9ac and H3K27me3 ChIPs, chromatin and antibodies were incubated overnight, rotating at 4ºC with 0.125-5 ug of specific antibody and samples were then incubated for 2 hours rotating at 4ºC with Dynabeads protein A for immunoprecipitation (Invitrogen) to recover the bound material. For H3K4me1, H3K4me2, H3K9me3, H4K20me1 and H3K27ac ChIPs, antibodies were coated to protein A magnetic beads for 2 hours at 4ºC prior to overnight incubation with chromatin. In all cases, beads were washed for 10' three times in 1 ml corresponding immunoprecipitation buffer without protease inhibitors, then washed once in 1 ml LiCl buffer (0.25 M LiCl, 0.5 % NP-40, 0.5 % sodium deoxycholate, 1 mM Na-EDTA, 10 mM Tris–HCl, pH 8.0) and finally washed twice in 1 ml of TE buffer (1 mM Na-EDTA, 10 mM Tris–HCl, pH 8.0). ChIPped material was incubated with DNase-free RNase at 50 ug/ml for 30' at 37ºC. Chromatin was reverse-crosslinked by adding SDS (0.5 % final concentration) and Proteinase K (500 ug/ml final concentration) and incubated overnight at 65ºC. ChIPped chromatin was then purified with Qiaquick PCR purification columns (Qiagen) following the manufacturer's instructions. ChIP libraries were prepared with 1-5 ng of DNA and using NebNext Ultra DNA library prep kit for Illumina (New England Biolabs) according to the manufacturer's protocol.