Novaseq sp kit

Total RNA was isolated from cells in triplicate separate cultures using RNeasy Mini Kit (Qiagen). Library preparation was performed using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD (Lexogen). Libraries were sequenced using a NovaSeq SP kit (Illumina) on a Novaseq instrument (Illumina).

Karpas-422 and K562 cells were crosslinked with 1% formaldehyde (Sigma-Aldrich) for 10 min and quenched with 125 mM glycine. Cells were lysed, sonicated (Branson sonifier, 0.7s on / 1.3s off, 5 min total on, 50% amplitude) and clarified by centrifugation. Lysates were incubated overnight at 4°C with the following antibodies: anti-EZH2 (Cell signaling Technology, #5246); anti-H3K27me3 (Cell signaling Technology, #9733S); anti-H3K36me3 (Cell signaling Technology, #9763). For quantitative ChIP, drosophila chromatin (Active Motif cat no. 08221011) and spike-in antibody (Active Motif cat no. AB27377370) were added to sonicated chromatin⁵⁹ . Dynabeads Protein G (Thermo Fisher) were added on the next day. Following incubation, the beads were isolated and washed. Immunoprecipitated chromatin was eluted, treated with RNase A and Proteinase K and purified by SPRI beads. To prepare ChIP-seq libraries, the chromatin was subjected to end-repair (End-It DNA End-Repair Kit, Lucigen), A-tailing (Klenow fragment, 3’−5’ exo-, NEB), ligation to barcoded adapters (KAPA) and PCR library amplification (KAPA), followed by SPRI-size selection and purification. Libraries were sequenced using a NovaSeq SP kit (Illumina) on a Novaseq instrument (Illumina).