GSM5284870: control-0h-rep3; Mus musculus; ChIP-Seq
RNA was extracted from cultured VSMCs using the Quick-RNA Micro Prep kit from ZymoResearch (Irvine, CA, #R1051), including optional DNase I treatment. mRNA was selected through poly-A isolation using Oligo d(T)25 beads (New England BioLabs, Ipswich, MA, #S1419S). Selected RNA was fragmented, followed by single strand cDNA synthesis using a SuperScript III First-Strand Synthesis System (ThermoFisher Scientific # 18080051), followed by second strand synthesis using DNA Polymerase I (Qiagen/Enzymatics, Beverly, MA, #P7050L). dsDNA ends were repaired with T4 DNA Polymerase (Enzymatics #P7080L). Barcode adapters (BIOO Scientific NEXTflex, Austin, TX, #514104) were ligated onto the ends of sequences using T4 DNA Ligase (Enzymatics #L-6030-HC-L) and samples were treated with Uracil DNA Glycosylase (UDG) (Enzymatics #G5010L). Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing. RNA was extracted from cultured VSMCs using the Quick-RNA Micro Prep kit from ZymoResearch (Irvine, CA, #R1051), including optional DNase I treatment. mRNA was selected through poly-A isolation using Oligo d(T)25 beads (New England BioLabs, Ipswich, MA, #S1419S). Selected RNA was fragmented, followed by single strand cDNA synthesis using a SuperScript III First-Strand Synthesis System (ThermoFisher Scientific # 18080051), followed by second strand synthesis using DNA Polymerase I (Qiagen/Enzymatics, Beverly, MA, #P7050L). dsDNA ends were repaired with T4 DNA Polymerase (Enzymatics #P7080L). Barcode adapters (BIOO Scientific NEXTflex, Austin, TX, #514104) were ligated onto the ends of sequences using T4 DNA Ligase (Enzymatics #L-6030-HC-L) and samples were treated with Uracil DNA Glycosylase (UDG) (Enzymatics #G5010L). Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing. cells were fixed at room temperature with 1% paraformaldehyde in PBS for 10 min, and then quenched with glycine. Between 3 and 5 million cells were used for each ChIP. Fixed lysates were sonicated using a BioRuptor Standard (Diagenode) Lysates were immunoprecipitated using antibodies bound to a 2:1 mixture of Protein A Dynabeads (Invitrogen #10002D) and Protein G Dynabeads (Invitrogen #10004D). Following immunopreciptation, crosslinking was reversed. dsDNA ends were repaired with T4 DNA Polymerase (Enzymatics #P7080L). Barcode adapters (BIOO Scientific NEXTflex, Austin, TX, #514104) were ligated onto the ends of sequences using T4 DNA Ligase (Enzymatics #L-6030-HC-L). Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing.