GSM6216482: BM-k4me3-SL4-3; Mus musculus; OTHER
Cells were resuspended in 500ul wash buffer (47.5ml H2O RNAse free, 1ml 1M HEPES pH 7.5, 1.5ml 5M NaCl, 3.6ul pure spermidine solution). Aliquot in a 0.5ml protein low-binding tubes (1 per antibody to be used). Following steps are performed on ice. Cells were pelleted at 600g for 3min and resuspended in 400 ul Wash Buffer 1 (wash buffer with extra 0.05% saponin (Sigma Aldrich), protease inhibitor cocktail (Sigma Aldrich), 4 ul/ml 0.5M EDTA) containing the primary antibody (1:100 dilution for H3K4me1, H3K9me3 and H3K27me3 and 1:200 dilution for H3K4me3, Saponin has to be prepared fresh every time as a 10% solution in PBS). Cells were incubated overnight at 4oC on a roller, before they were washed once with 500 ul Wash Buffer 2 (wash buffer with extra 0.05% saponin, protease inhibitor). Afterwards cells were resuspended in 500 ul Wash Buffer 2 containing PaMN (3ng/ml) and incubated for 1h at 4C on a roller. Finally, cells were washed an additional 2 times with 500 ul Wash Buffer 2 before passing it through a 70um cell strainer (Corning, 431751) and sorting them on a JAZZ FACS machine into 384 well plates containing 50nl Wash buffer 3 (Wash buffer containing 0,05% saponin) and 5ul sterile filtered mineral oil (Sigma Aldrich) per well. Small volumes were distributed using a Nanodrop II system (Innovadyme). 50nl of Wash Buffer 3, containing 4uM CaCl2, were added per cell to induce Protein A-MN mediated chromatin digestion that was performed for 30min on ice. Afterwards the reaction was stopped by adding 100nl of a stop solution containing 40mM EGTA (chelates Ca2+ and stops MN), 1.5% NP40 and 10ul 2mg/ml proteinase k (Ambion). Chromatin is subsequently released and PaMN permanently destroyed by proteinase k digestion at 65C for 6h followed by 80C for 20min to heat inactivate proteinase k. Afterwards plates can be stored at -20C until further processing. DNA fragments are further blunt ended using Klenow large fragment (NEB) and T4 PNK (NEB) in the presence of dNTPs. Afterwards nonintegrated dNTPs are removed using schrimp alkaline phosphatase (NEB) to increase A-tail efficiency. Subsequently blunt fragments are A-Tailed using AmpliTaq™ 360 DNA Polymerase (Thermofisher) in the presence of fresh dATP and T4 PNK (NEB). Next fragments are ligated to T-tail adaptors containing 3 bp UMIs and an 8bp cell barcode (final concentration 9nM from IDT) for 16h at 16C using T4-ligase (NEB). Adaptors were added with a Mosquito HTS (ttp labtech). Ligation products were pooled by centrifugation and cleaned using Ampure XP beads at a bead to sample ratio of 0.8 (Beckman Coulter). The cleaned DNA is then linear amplified by invitro transcription with components of MessageAmp II (Invitrogen) for 16h at 37C. The produced RNA is purified 1-3 times depending on Adaptor dimer contamination using RNA Clean XP beads (Beckman Coulter) at 0.8 beads to sample ratio, followed by RNA fragmentation for 2.5 min at 94C. After another bead cleanup, 20% of the RNA is reverse transcribed with Superscript II (NEB) and PCR amplified to add the Illumina smallRNA barcodes and handles with NEBNext Ultra II Q5 Master Mix. PCR cycles depended on the abundance of the histone modification assayed and the number of RNA cleanup cycles after IVT needed (8-10 for H3K9me3 and H3K27me3 10-12 for H3K4me1 and 12-15 for H3K4me3).