title: GSM8128003: RNAseq_diff_dTAG_24H_RA_24H_rep2; Mus musculus; RNA-Seq
library_construction_protocol: ChIP-seq: Chromatin was cross-linked using 1% FA final concentration. Chromatin was sheared on a Bioruptor Plus sonication instrument and immunoprecipitated by different antibodies. DNA was purified using the MiniElute PCR purification kit from Qiagen. TT-seq: Libraries for TTchem-seq were prepared following a published protocol (Gregersen et al., 2020). Cells were labelled with 2 mM of the uridine analog 4-thiouridine (4sU) for 10 min. Total RNA was isolated and fragmented. The 4sU-biotin labelled RNA was enriched using µMacs Streptavidin Kit (Miltenyi). Hi-C: Hi-C data were generated using as a previous published protocol (Rao et al., 2014) with minor modifications (Haarhuis et al., 2017). Cells were harvested and crosslinked using 2% formaldehyde. Crosslinked DNA was digested in nucleus using MboI (NEB), and biotinylated nucleotides were incorporated at the restriction overhangs and joined by blunt-end ligation. The ligated DNA was enriched in a streptavidin pull-down 4C-seq: 4C-seq was performed as previously described (Geeven et al., 2018; van de Werken et al., 2012) using a two-step PCR method for indexing described first in (Haarhuis et al., 2017). MboI (NEB) was used as the first restriction enzyme and Csp6I (NEB) as the second restriction enzyme. ATAC-seq: Cells were washed in cold PBS and subjected to lysis using a 2x lysis buffer. Subsequently, the cells were centrifuged, and the resulting pellet was treated with 2xTD buffer and 2 uL of transposon mix. RNA-seq: RNA was first isolated using standard RNA isolation procedure from Qiagen RNeasy Mini Kit (Qiagen), including a DNaseI treatment. ChIP-seq: Libraries were prepared following the KAPA HTP Library Preparation Kit (Roche). TT-seq: Libraries were prepared using KAPA RNA HyperPrep and KAPA Dual-Indexed Adapter kits (Roche) using dual indexing adapters. Hi-C: Hi-C libraries were prepared using a standard end-repair and A-tailing method. 4C-seq: 4C-seq libraries were prepared using a two-step PCR method for indexing described first in (Haarhuis et al., 2017). ATAC-seq: PCR amplification was conducted using KAPA HiFi HotStart ReadyMix (Roche). Fragments ranging from 200 to 700 bp were purified utilising AMPure XP beads (Beckman Coulter). RNA-seq: Libraries were prepared following the TruSeq Stranded mRNA kit (Illumina) with TruSeq RNA Single Indexes set A (Illumina).