title: GSM7673389: MAbID library Pol2Ser5P (H3+H3K27me3+H3K36me3+Pol2Ser5P); Homo sapiens; OTHER
library_construction_protocol: Cells were harvested (~10 x 106 cells) and washed once with PBS. All centrifugation steps were at 200 g for 4 minutes at 4 °C. Cells were fixated in 1% formaldehyde (Sigma, F8875) in PBS for 5 minutes, before quenching the reaction with 125 mM final concentration of glycine (Sigma, 50046) and placing the cells on ice. All subsequent steps were performed on ice or at 4⁰C. Cells were washed three times with PBS before resuspension in Wash buffer 1 (20mM HEPES pH 7.5 (Gibco, 15630-056), 150 mM NaCl, 66.6 µg/mL Spermidine (Sigma, S2626), 1X cOmplete™ protease inhibitor cocktail (Roche, 11697498001), 0.05% Saponin (Sigma, 47036), 2mM EDTA) and transferred to a 1.5 mL protein LoBind Eppendorf tube (Eppendorf, EP0030108116-100EA). Cells were permeabilized for 30 minutes at 4 ⁰C on a tube roller. Bovine Serum Albumin (BSA, Sigma, A2153) was added to 5 mg/mL final concentration and incubated for another 60 minutes at 4 ⁰C on a tube roller. Permeabilized nuclei were used for antibody incubation. Samples ligated with unique SBC adapters were pooled, either 2-4 1000 nuclei samples or a full 384-well plate were pooled for combined in vitro transcription (IVT). To reduce batch effects, controls (secondary antibody-conjugates without primary antibody incubation) were pooled with their corresponding samples whenever possible. For 384-well plates, mineral oil was removed by spinning the sample for 2 minutes at 2000 g and transferring the liquid phase to a clean tube, which was repeated three times. After pooling, samples were incubated for 10 minutes with 1.0 volume CleanNGS magnetic beads (CleanNA, CPCR-0050), diluted 1:4 to 1:10 in bead binding buffer (20% PEG 8000, 2.5 M NaCl, 10 mM Tris–HCl, 1 mM EDTA, 0.05% Tween 20, pH 8.0 at 25 °C). The bead dilution ratio depended on the total volume, 1:4 for 1000 nuclei samples and 1:10 for a full 384-well plate. Samples were placed on a magnetic rack (DynaMag™-2, ThermoFisher, 12321D) to collect beads on the side of the tube. Beads were washed two times with 80% ethanol and briefly allowed to dry before resuspending in 8 µL water. In vitro transcription was performed by adding 12 µL IVT mix from the MEGAScript T7 kit (Invitrogen, AM1334) for 14 hours at 37 ⁰C before holding at 4 ⁰C. Library preparation was subsequently performed as described previously 13,46, using 5 µL of aRNA and 8 to 11 PCR cycles, depending on the aRNA yield. Purified aRNA from different IVT reactions (with unique SBC adapters) can be pooled before proceeding with cDNA synthesis to reduce batch effects. Libraries were run on the Illumina NextSeq500 platform with high output 1x75 bp, the Illumina NextSeq2000 platform with high output 1x100 bp or the Illumina NextSeq2000 platform with high output 2x100 bp.