title: GSM9000894: Valid_mESC_RNA-ESC2EpiLC_WT-0h-1; Mus musculus; RNA-Seq
library_name: GSM9000894
library_construction_protocol: RNA-seq: RNA was extracted from ESCs and EB spheroids using TRIzol Reagent (Invitrogen) followed by chloroform extraction. RNA was then precipitated using isopropanol with the addition of 1/10 volume of 3M NaAC and 2μL glycogen to the aqueous phase. After two washes with 75% ethanol, RNA was resuspended in nuclease-free water. CUT&Tag: Cells were bound to ConA beads and incubated with primary antibody overnight, followed by secondary antibody incubation. Tagmentation was performed using pG-Tn5, and DNA was purified using VAHTS beads and amplified by PCR. Libraries were prepared using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme Biotech, TD904). ULI-NChIP-seq was performed using 200-500 cells/ sample, with two or three biological replicates per group. Isolated cells were washed with PBS/BSA, and the ULI-NChIP procedure was performed as previously described using 1μg of a histone H3K27me3 or H3K4me3 antibody per immunoprecipitation reaction. XChIP-seq: ESCs/sample were used (2 replicates) for ZBTB17 XChIP-seq. Cells were crosslinked (1% formaldehyde), quenched (glycine), lysed, and sonicated (peak power 75, duty factor 20, cycles 200, 10 min). Chromatin was immunoprecipitated using ZBTB17 antibody and protein A/G beads. DNA was eluted, purified, and precipitated. Libraries were prepared using the KAPA Hyper Prep kit. reChIP-seq: ESCs and EPILCs were processed using the same crosslinking, lysis, and sonication steps as the XChIP-seq protocol. Sequential ChIP was performed using the reChIP-IT kit (Active Motif, 53106). RNA-seq and ChIP-seq: Libraries were prepared using the KAPA Hyper Prep Kit. Sequenced on Illumina Novaseq. CUT&Tag: Libraries were prepared using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme Biotech, TD904).
identifiers.namespace: GEO
identifiers.id: GSM9000894_r1