GSM9241836: TTseq_NA18983_rep2; Homo sapiens; OTHER
10X linked-reads: Linked-reads data generation was outsourced to NGX Bio. For each sample, 3 million cells were harvested in PBS and the frozen cell pellets were sent to NGX Bio for processing. The resulting high molecular weight DNA was extracted using the MagAttract HMW DNA Extraction Kit (Qiagen). The quality of the DNA was assessed using the TapeStation system (Agilent Technologies). 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. 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. 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). 10X linked-reads: Libraries were prepared using the 10X Genomics GemCode platform with reagents from 10X Genomics and the KAPA Hyper Prep Kit (Roche). Hi-C: Hi-C libraries were prepared using a standard end-repair and A-tailing method. 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). TT-seq: Libraries were prepared using KAPA RNA HyperPrep and KAPA Dual-Indexed Adapter kits (Roche) using dual indexing adapters.