title: GSM8657175: iALL2 H3K4me1; Homo sapiens; ChIP-Seq
library_construction_protocol: TOPMentation Briefly, protein A-coupled magnetic beads (10 µl) were incubated with 1 µl of the appropriate antibody for 4 h with rotation at 4 °C in PBS with 0.5% BSA and 1× protease inhibitor cocktail. The antibodies used are detailed in Supplementary Table 1. Cells were single-fixed (1% formaldehyde for 10 min) for histone modifications or double-fixed (2 mM disuccinimidyl glutarate for 30 min, then 1% formaldehyde for 30 min) for transcription/chromatin factors. Fixed samples were lysed in 120 µl of lysis buffer (50 mM Tris-HCl pH 8.0, 0.5% SDS, and 10 mM EDTA, 1× protease inhibitor cocktail) and sonicated using a Covaris ME220 (Woburn, MA) to generate 200–300 bp fragments. The sonicated chromatin was then incubated with Triton-X100 at a final concentration of 1% for 10 min at room temperature to neutralize the SDS in the lysis buffer. The chromatin was pre-cleared for 30 min at 4 °C with rotation using 5 µl protein A-coupled beads to reduce non-specific binding. The antibody-incubated beads were washed in 150 µl Binding Wash Buffer (PBS with 0.5% FCS), and the pre-cleared chromatin was then added to the antibody-coated beads before incubation overnight at 4 °C with rotation. Immunoprecipitated chromatin was washed three times with RIPA buffer (50 mM HEPES-KOH pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% Na deoxycholate). Beads were transferred to a second tube between the first and second wash to remove non-immunoprecipitated fragments that adhered to the tube. The beads were then washed once with Tris-EDTA and once with 10 mM Tris-HCl pH 8.0. The chromatin was then tagmented by resuspending beads in 29 µl Tagmentation Buffer (10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 10% dimethylformamide) and adding 1 µl of transposase (Illumina). Samples were incubated at either 37 °C or 50oC for 5 min 5 mins (dependent on the activity of the transposase lot), and the reaction was terminated by adding 150 µl RIPA buffer. Beads were washed with 10 mM Tris-HCl pH 8.0 to remove any detergent and resuspended in 22.5 µl ddH2O. In order to amplify the tagmented chromatin, 25 µl NEBNext Ultra II Q5 Master Mix (NEB) and indexed amplification primers (125 nM final concentration) were added, and libraries were prepared using the following thermal profile: 72 °C 5 min, 95 °C 5 min, (98 °C 10 s, 63 °C 30 s, 72 °C 3 min) × 12 cycles, 72 °C 5 min. Library clean-up was performed using Agencourt AMPure XP beads at a 1:1 ratio. Samples were sequenced by paired-end sequencing on a NextSeq 500 or on a NovaSeq6000 S4 flow cell.