RNA in situ conformation sequencing (RIC-seq) in naive and primed mouse embryonic stem cells
RNA in situ conformation sequencing (RIC-seq) in naive and primed mouse embryonic stem cells
Naive (nPSCs) IDG3.2 G9 (p20) and primed (pPSCs) IDG3.2 G9 (p13) were used for the experiment; two confluent 10 cm plates were used per sample and triplicates were used per cell state.  Naive cells (nPSCs) were maintained in 2i media (PMID: 18497825), fed every day and split every 2-3 days using Accutase (A6964, Sigma). Primed cells (pPSCs) were obtained previously from naive state and maintained in EpiSC/primed medium (N2B27 basal medium with ActA 20 ng/ml, bFGF 12 ng/mL, IWP2 2 µM) on cell culture dishes (TPP) coated with Geltrex (A1413302, Gibco), fed every day and split every 2-3 days using Accutase (A6964, Sigma).   Cells were washed with PBS and dissociated with Accutase before crosslinking with freshly prepared 0.5 mg/ml DSS (21655, Thermo Fisher Scientific) at RT for 30 min. The reaction was quenched using 20 mM Tris (pH7.5) for 15 minutes at room temperature and cells were pelleted by centrifugation at 3500 rpm for 5 minutes at 4°C (all centrifugations were done at this conditions). After pelleting the cells, they were washed with PBS and permeabilised on ice for 15 minutes. Cells were then treated with 6U MNase at 37°C for 10 minutes, then treated with FastAP and finally followed by pCp-biotin ligation (Thermo Scientific, 20160). Finally, another FastAP and PNK treatments were done before proximity ligation using T4 RNA ligase overnight. Cells were lysed using proteinase K and Turbo DNase; after adding TRIzol-LS to the lysate, samples were stored at -80°C before RNA extraction. TRIzol samples were brought to room temperature and heated for 10 minutes for 55°C (following semi-extractability protocol, PMID: 28404604) before adding chloroform and extracting RNA from the aqueous phase (according to manufacturer's instructions). After treating RNA with TURBO DNase, 21 μg of RNA was fragmented using 5x First Strand Synthesis Buffer, and mixed with the MyOne Streptavidin C1 beads to pulldown biotinylated RNA (30 minutes at room temperature). Eluted RNA was isolated using phenol:chloroform extraction. Biotinylated RNA was subjected to 3'end dephosphorylation using PNK and FastAP before 3'end adapter ligation. Then, reverse transcription was done using Superscript IV and custom RT primer. Following cDNA cleanup, 5' cDNA adapter was ligated and the samples were loaded onto 6% TBE-Urea gel. cDNA longer than 200 bp was excised from the gel and extracted before final PCR reaction with P5/P7 primers. Ribosomal RNA contaminants were removed from the final library using Ribocutter which utilises Cas9-guided rRNA depletion (https://doi.org/10.1101/2021.07.14.451473).