GSM5885023: rS2: MEF_wt+non_targeting siRNA rep 2 [RPF]; Mus musculus... - SRA

SRX14127408: GSM5885023: rS2: MEF_wt+non_targeting siRNA rep 2 [RPF]; Mus musculus; RNA-Seq
1 ILLUMINA (NextSeq 550) run: 84.4M spots, 6.4G bases, 2.1Gb downloads

External Id: GSM5885023_r1

Submitted by: Deihl Lab, Department of Biochemistry, Case Western Reserve University

Study: Ribosome profiling of Murine Embryonic Fibroblasts in the context of different Fbxo4 and hnRNPK status.

PRJNA805181 • SRP359195 • All experiments • All runs

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Background: SCF-Fbxo4 is suspected to regulate activity of RNA binding protein hnRNPK by non-degradive ubiquitylation. Research strategy: To determine the genomic output of SCF-Fbxo4 - hnRNPK interplay, murine embryonic fibroblasts (MEFs) with different status of hnRNPK/Fbxo4 were subjected to Ribosome profiling. Sequencing was performed on total RNA and RNA protected by ribosomes for each condition. Overall design: Applying RNAi strategy hnRNPK was depleted in either wild type MEFs or MEFs Fbxo(-/-). MEFs conditioned in 4 different ways: wt- control (I), Fbxo4 knock-out (II), Fbxo4 knock-out + hnRNPK RNAi knock-down (III) and RNAi hnRNPK knock-down only (IV) were subjected to sequencing of total RNA and Ribosome protected fragment.

Sample: rS2: MEF_wt+non_targeting siRNA rep 2 [RPF]

SAMN25829770 • SRS11954503 • All experiments • All runs

Organism: Mus musculus

Library:

Name: GSM5885023

Instrument: NextSeq 550

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: SINGLE

Construction protocol: Cell extract was treated with MNase, CaCl2 (5mM final concentration), and Turbo DNAse I and incubated at 25°C for 30 minutes; the digestion was stopped with SUPERase-inhibitor and placing on ice. The cell extract was loaded onto a 15%-45% sucrose gradient to separate the polysomes by ultra-centrifugation. The relevant fractions corresponding to the 80S monosome were collected and precipitated using ethanol and the RNA was isolated Total RNA was isolated from each sample and subjected to the Turbo DNase I digestion and then RNA-seq libraries. The ribosome-protected fragments were subjected to denaturing polyacrylamide-mediated gel electrophoresis and were gel-extracted based on size (26-mers to 34-mers) followed by rRNA depletion by RiboMinus kit (Ambion) For total RNA sequencing, libraries were constructed using Illumina's TruSeq Stranded Total RNA kit. The ribosome protected fragment library was generated using NEXT-Flex smRNA-Seq Kit v3 (Perkin Elmer)

Runs: 1 run, 84.4M spots, 6.4G bases, 2.1Gb

Run	# of Spots	# of Bases	Size	Published
SRR17971140	84,429,864	6.4G	2.1Gb	2022-09-21

ID:: 19919486

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