title: GSM6541900: shPALI1_PALI1 CUT&RUN_Rep2; Homo sapiens; OTHER
library_construction_protocol: For SUZ12 and G9a ChIP-Seq, Chromatin in nuclear fraction were sheared to 200-500bp using an Covaris M220 Focused-ultrasonicator, Lysates were clarified from sonicated nuclei and protein-DNA complexes were immunoprecipitated using the indicated antibody. DNA was then reverse crosslinked from protein and purified.  For H3K27me3, H3K9me2 CUT&Tag and PALI1 CUT&RUN, 0.5x10^6 LNCaP cells bound to Concanavalin A-coated beads were incubated with indicated primary antibodies and secondary antibody. pA-Tn5 was used to induce DNA tagmentation in CUT&Tag while pA-MNase was used to digest chromatin/DNA bound by the antibodies-protein complex in CUT&RUN, followed by Proteinase K digestion and PCI extraction. For CUT&Tag, the ethanol precipitated DNA was immediately subjected to PCR amplification by Universal and barcoded i7 primer using NEBNext HiFi 2x PCR Master mix. The amplified library was purified by using Agencourt AMPure XP beads without size selection. For ChIP-Seq and CUT&RUN, ChIP-seq libraries were prepared from 3-5ng ChIPped DNA while CUT&RUN libraries were prepared from 15-20ng DNA using NEBNext® Ultra™ II DNA Library Prep Kit (NEB, E7645S), according to the manufacturer's instructions. Postamplification libraries were size selected at 250–450 bp (ChIP-Seq) and 160-320bp (CUT&RUN) in length using Agencourt AMPure XP beads from Beckman Coulter and were quantified using the Library Quantification Kit from Illiumina (Kapa Biosystems, KK4603). All CUT&Tag, CUT&RUN and ChIP-Seq Libraries were pooled to a final concentration of 10nM and sequenced single-end using the Illumina HiSeq 4000.