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SRX24387165: GSM8239953: wt_2_up; Homo sapiens; OTHER
2 ILLUMINA (Illumina HiSeq 4000) runs: 83.3M spots, 4.2G bases, 1.5Gb downloads

External Id: GSM8239953_r1
Submitted by: Biochemistry and Biophysics, UCSF
Study: CRISPRi Screen for Investigating the RORC RNA Switch Mechanisms in Jurkat Cells
show Abstracthide Abstract
RNA structural switches are key regulators of gene expression in bacteria, yet their characterization in Metazoa remains limited. Here we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3'UTR of the RORC transcript. In vivo DMS-MaPseq, coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated mRNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally-driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape. Overall design: To investigate how the RORC RNA switch influences gene expression at the molecular level, we performed genome-wide CRISPRi screens in Jurkat T cells using two eGFP reporter constructs: one with the native RORC switch and another with the 77-GA mutation that favors conformation 1.
Sample: wt_2_up
SAMN41105992 • SRS21143220 • All experiments • All runs
Organism: Homo sapiens
Library:
Name: GSM8239953
Instrument: Illumina HiSeq 4000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: On Day 11, cells were collected and sorted using the BD FACSAria™ Fusion cell sorter. Sorting criteria included the top and bottom 25% of cells based on the ratio of GFP to mCherry fluorescence intensity. The CRISPRi screen was conducted under two conditions using cells with either a reference RORC element - GFP reporter or a mutated 77-AG RORC element - GFP reporter, with each condition tested in duplicate. After sorting, genomic DNA was harvested (Macherey-Nagel Midi Prep kit) and amplified using NEB Next Ultra II Q5 master mix and primers containing TruSeq Indexes for NGS analysis. Sample libraries were prepared and sequenced on a HiSeq 4000. The library preparation for CRISPRi/a-v2 involves amplifying sgRNA sequences, which are then sequenced on an Illumina HiSeq 4000 system. The library includes TruSeq indices for demultiplexing post-sequencing. The PCR amplification incorporates Illumina adapters and a unique sample index, facilitating the sequencing of pooled samples in a single sequencing run with a phiX spike-in of 10% to enhance sequence diversity and read accuracy.
Runs: 2 runs, 83.3M spots, 4.2G bases, 1.5Gb
Run# of Spots# of BasesSizePublished
SRR2882426841,707,4162.1G769.8Mb2024-07-01
SRR2882426941,569,0852.1G761.2Mb2024-07-01

ID:
32702146

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