GSM3350469: H3K27me3_A1N2_mL_mD_2; Mus musculus; ChIP-Seq
Chromatin: cells were resuspended in 3 ml PBS and crosslinked for 10 min at room temperature with 1% formaldehyde (Sigma F8775). Crosslinking was stopped with 0.125 mM glycine for 5 min at room temperature. 44iN cells (3.107 ) used for TF binding profiling were crosslinked in 3 ml of freshly prepared PBS-DSG 7/17 2 mM at pH 7.0 (Sigma, 80424-5 mg) for 50 min at room temperature with occasional shaking. After pelleting and washing in PBS, cells were incubated for 10 min in 3 ml PBS 1% formaldehyde (Sigma F8775), quenched with 0.125 MM glycine. After fixation, cells were pelleted, washed twice with ice-cold PBS and resuspended in 6 ml of swelling buffer (25 mM Hepes pH 7.95, 10 mM KCl, 10 mM EDTA) freshly supplemented with 1X protease inhibitor cocktail (PIC-Roche, 04 693 116 001) and 0.5% NP-40. After 30 min on ice with occasional shaking, the suspension was centrifuged and resuspended in 450 μl of TSE150 (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl pH8, 150 mM NaCl) buffer, freshly supplemented with 1X PIC. Samples were split in 3 (150µL) and sonicated in 1.5 mL tubes (Diagenode) using a Bioruptor Pico (Diagenode) for 7 cycles divided into 30 s ON - 30 s OFF sub-cycles at maximum power, in circulating ice-cold water. After centrifugation (10 min, full speed, 4 °C), the supernatant was stored at −80 °C. Five microlitres was used to quantify the chromatin concentration and check DNA size (typically 200–500 bp). ChIP-seq: Chiped DNA was end repair in a total volume of 50µL (sample 37.5 µL, 10mM dNTPs 2 µL, NEB T4 ligase buffer 5 µL, NEB T4 polymerase 2.5 µL, NEB Klenow polymerase 0.5 µL, NEB T4 PNK 2.5 µL) and incubated for 30min at 20°C. After DNA purification (see below), A-tailing was subsequently performed in a total volume of 25 µL (sample 20 µL, NEB Buffer 2 2.5 µL, 5mM dATP 1 µL, NEB Klenow 3'-5' exo minus 1.5 µL) at 37°C for 30min. Illumina TruSeq adapters were used for libraries indexing, ordered from IDT Company with 5' phosphate modification. Illumina adapters' compatibility was checked with the online tool checkmyindex (checkmyindex.pasteur.fr) for multiplexing. Truseq adapters were annealed with Illumina Universal adapter at 20µM each in 1X NEB Buffer 2 on a TM 100 Thermal Cycler (Biorad). Adapters ligation was performed in a total volume of 25 µL (sample 19.25 µL, NEB 10x T4 ligase buffer 2.5 µL, 0.2µM adapter 1.25 µL, NEB T4 ligase 2 µL) at 16°C overnight. After DNA purification, DNA was amplified in a total volume of 50 µL (sample 19.5 µL, Pico green 1 µL – 9/17 1:10 in water; Life Technologies P7589 –, 25 µL of KAPA HiFi HOTSTART Ready mix – NC0295239 –, PCR 1.0 10µM 1 µL, PCR 2.0, 10µM 1 µL) on a LightCycler 480 (Roche). PCR primers are listed in Table S4. Any sample reaching an absolute fluorescence value of 6 was taken out from the plate at the end of the last extension. Any library requiring more than 16 cycles of amplification to reach this level was discarded and reprepared. DNA was finally purified and the concentration was measured with a Qubit 4 Fluorometer (Thermo Q33226). Libraries quality check and size estimation were then performed on an Agilent 2200 Tape Station with High Sensitivity D5000 ScreenTape (Agilent Technologies, 5067-5592) and High Sensitivity D1000 Reagents (Agilent Technologies, 5067-5585) using 1-2ng of material. Libraries were subsequently adjusted to equimolar concentration of 2nM according to fragments average size and concentration prior mixing them for subsequent sequencing on the HiSeq 2500 sequencer (Illumina) in 65 bases V4 single-end mode
GEO Accession
GSM3350469