biostudies-arrayexpressTajda KlobučarMus musculushttps://github.com/amchakra/toscaUMI-tools(https://umi-tools.readthedocs.io/), Cutadapt (https://cutadapt.readthedocs.io/), BBMerge (https://jgi.doe.gov/data-and-tools/software-tools/bbtools/bb-tools-user-guide/bbmerge-guide/),Tosca (https://github.com/amchakra/tosca)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14764RNA in situ conformation sequencing (RIC-seq; PMID: 32499643) data at two distinct pluripotent stage of mouse embryonic stem cells (naive - nPSCs and primed - pPSCs).biostudies-arrayexpressNucleic Acid Extraction - 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.Sequencing - Libraries were sequenced as paired-end 150 bp reads on NovaSeq at Clinical Institute of Special Laboratory Diagnostics, University Children's Hospital at Ljubljana University Medical Centre.Library Construction - 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).Sample Collection - 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.Growth Protocol - 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).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The hybrid read files were automatically generated by the Tosca v1.0.0 analysis pipeline.Sequence Alignment - The raw FASTQ files were processed as follows: UMIs (located at the start of R2) were extracted and moved to read headers using UMI-tools (UMI pattern: 'NNNCCCCNNN'). Sequencing adapters ('AGATCGGAAGAG') were trimmed, and low-quality bases were removed from the 3' end using Cutadapt, retaining only reads with a minimum length of 16 nucleotides. Read mates were then merged using BBMerge with default settings. The resulting merged FASTQ files were analysed with Tosca v1.0.0 to identify hybrid reads generated by RNA proximity ligation. For hybrid identification and annotation, the mouse reference genome (GRCm39, GENCODE release M27) was used.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of total RNAMus musculusTajda KlobučarIra IosubMiha ModicfalseRNA in situ conformation sequencing (RIC-seq) in naive and primed mouse embryonic stem cellsRNA in situ conformation sequencing (RIC-seq; PMID: 32499643) data at two distinct pluripotent stage of mouse embryonic stem cells (naive - nPSCs and primed - pPSCs).2025-09-16T00:00:00Z2025-09-17T01:02:48.569Z2025-01-20T22:28:40.211ZE-MTAB-14764ERP168502EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184