GSM6430050: ChIP_H3K4me1_D50_r2; Drosophila melanogaster; ChIP-Seq
Isolate D10 to D50 fly heads (50mg) for each biological replicate. Fly heads were dounced 30 times with pestle A (Sigma) in 5 ml of A1 buffer (60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 15 mM HEPES pH 7.6, 0.5% NP-40, 0.5 mM DTT, 0.5 mM PMSF and protease inhibitors cocktail). Samples were filtered through MIRA cloth, and the lysate was incubated on ice for 15min. Samples was then centrifuged at 4000g for 5min at 4°C. After three additional rounds of A1 buffer wash and centrifugation, nuclear pellet was resuspended and dounced with pestle B (Sigma) in 3.3 ml of RIPA buffer (10 mM Tris pH 8.0, 1 mM EDTA, 140 mM NaCl, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate, 0.5 mM DTT, 0.5 mM PMSF, protease inhibitors cocktail). 3.3 µl of 3M CaCl2 was added to 3.3 ml of nuclear lysate and incubated at 37°C for 3min. 5µl of MNase (i-DNA Biotech, Cat# LS004798) was then added to samples and incubated at 37°C for 15 min. MNase reaction was terminated by adding STOP buffer (5µM EDTA/EGTA). Samples were briefly sonicated (10sec ON, 50sec OFF, 5 times), followed by 10 min max speed centrifugation at 4°C. The supernatant was collected as the chromatin extract and 80 µl was kept as input. 5ul of H3K4me1 (Abcam, ab8895) or H3K27ac (Abcam, ab4729) antibody was added to remaining chromatin extract and incubate in 4°C overnight. On the following day, 20 μl of pre-blocked (0.5% BSA in PBS) Protein-G Dynabeads™ (Lifetech, Cat# 10003D) was added to the chromatin extract. Following 4 hours of incubation at 4°C, DNA-antibody-beads complex was isolated using magnetic stand. The beads complex was washed with RIPA buffer for 5 times, LiCl buffer (10 mM Tris pH 8, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) once and Tris-EDTA buffer (10 mM Tris pH 8, 1 mM EDTA) once. The DNA fragments were eluted from the beads with 500 μl elution buffer (1 M NaHCO3, 0.5% SDS), while input was topped up to 420 µl with elution buffer and samples were incubated at 37°C overnight. On the next day, samples were treated with RNase A (20 μg/ml, 1 hr at 37°C) and proteinase K (40 μg/ml, 2 hr at 37°C). DNA was extracted by phenol/chloroform and ethanol precipitation. ChIP-sequencing was carried at BGI (Hong Kong) according to company's protocol. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified using KAPA library amplification kit (KK2702) with Illumina primers for 11 cycles and library fragments of ~200-500 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq 4000.