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SRX997569: GSM1659716: chd1_rep3_fRIP-seq; Homo sapiens; OTHER
4 ILLUMINA (Illumina HiSeq 2500) runs: 29M spots, 1.8G bases, 869.3Mb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Widespread RNA Binding by Chromatin Associated Proteins
show Abstracthide Abstract
Recent evidence suggests that RNA interaction can regulate the activity and localization of DNA binding proteins, particularly chromatin-associated proteins. However, it is unknown if these observations are specialized instances for a few key RNAs and chromatin factors in specific contexts, or a general mechanism underlying the establishment of chromatin state and regulation of gene expression. Here, we introduce formaldehyde RNA ImmunoPrecipitation (fRIP-Seq), a sensitive method for cataloging RNA-protein interactions, to survey the RNA associated with a panel of 24 chromatin-associated and traditional RNA binding proteins. For each protein that reproducibly bound measurable quantities of bulk RNA (90% of the panel), we detected enrichment for hundreds to thousands of both noncoding and mRNA transcripts. We found that the enriched sets of RNA share biochemical, functional, and epigenetic properties. Thus, these data provide strong evidence that non-random RNA association is a common feature across diverse classes of chromatin modifying complexes. Overall design: RNA associated with 24 different proteins using fRIP was compared to total RNA-seq
Sample: chd1_rep3_fRIP-seq
SAMN03487448 • SRS911838 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: We re-suspended frozen pellets in 1 mL of RIPA lysis buffer (50mM Tris (pH 8), 150 mM KCl, 0.1% SDS, 1% Triton-X, 5 mM EDTA, 0.5% sodium deoxycholate,0.5 mM DTT (add fresh) + protease inhibitor cocktail (Thermo Scientific, PI-87785) + 100 U/ml RNaseOUTTM  (Life Technologies , 10777-019)). We incubated cells at 4°C for 10 minutes before lysing on a Branson® digital sonifier using 10% amplitude for 0.7 seconds on and 1.3 seconds off at 30 second intervals for a total of 90 seconds. We used chilled tube holders and swapped them out between shearing runs to reduce temperature elevation. After lysis, we spun the lysate at 4°C max speed for 10 minutes.  We collected supernatant and diluted by adding equal volume of fRIP binding/wash buffer (150 mM KCl, 25 mM Tris (pH 7.5), 5 mM EDTA, 0.5% NP-40, 0.5 mM DTT (add fresh), 1X PIC (add fresh), 100 U/mL RNaseOUT  (add fresh)). At this point, we removed 50 μl of lysate for input sample and stored at -20°C for later RNA purification and library construction. After dilution, we clarified the lysate by passage through a 0.45 μM syringe filter. We then “pre-cleared” filtered lysate by incubating with Dynabeads® Protein G (Life Technologies cat#10004D) at a concentration of 25 μl of beads per 5 million cells for 30 minutes at 4°C with slow rotation. We flash froze pre-cleared lysate in 1 mL aliquots of ~5 million cells and stored it at -80 °C. For fRIP, we thawed lysate on ice and added 6 μg of HuR antibody (Santa Cruz, sc-5483). After addition of antibody, we rotated lysate at 4°C for 2 hours before adding 50 μl of Dynabeads® Protein G. We rotated beads and lysate at 4°C for 1 hour before washing 2X with 1 mL of fRIP binding/washing buffer + 1X PIC and 100 U/mL RNaseOUT. After the final wash, we removed the supernatant and froze and stored the beads at -20°C. We re-suspended the frozen beads in 56 μl of RNase-free water and added 33uL of 3X reverse-crosslinking buffer (3X PBS (without Mg or Ca), 6% N-Lauroyl Sarcosine, 30 mM EDTA, 15 mM DTT (add fresh)), 10 μl of Proteinase K (Life Technologies, cat #AM9516), and 1 μl of RNaseOUT to both the re-suspended beads and input sample. We performed protein degradation and reverse-crosslinking for 1 hour at 42°C, then another 1 hour at 55°C. We added beads and reaction buffer to 1 mL of TriZol (Life Technologies, 15596-026). After agitation, we added 200 μl of chloroform followed by ~15 seconds of vigorous agitation and a 20 minute microcentrifuge spin at 4°C max speed. We collected the aqueous layer, added it to 750 μl of ethanol + 1 μl GlycoBlue™, and ran it over a Qiagen RNeasy® min-elute column (Qiagen, cat #74204). We extracted RNA using the buffer RWT/3X isopropanol modification detailed in “Appendix B: Optional On-Column DNase Digestion…” of the Qiagen miRNeasy® Mini Handbook. We eluted RNA in 15μl of RNase-free water. to remove ribosomal RNA, we fed ≥ 70 ng of input and fRIP RNA into the Ribo-Zero™ Magnetic Gold Kit (Epicentre, cat #MRZG12324) followed by a cleanup using Agencourt RNAClean XP beads (Beckman Coulter, cat #A63987) and elution with 19.5 uL of Elute, Prime, Fragment mix from the TruSeq RNA Sample Preparation Kit (Illumina, cat #RS-122-2001). We performed library construction per the vendor’s instructions, starting with the “Incubate RFP” step. We pooled the resulting cDNA libraries and subjected them to paired-end sequencing on an Illumina HiSeq 2500 at a depth of 31 base pairs per read.
Experiment attributes:
GEO Accession: GSM1659716
Links:
External link:
Runs: 4 runs, 29M spots, 1.8G bases, 869.3Mb
Run# of Spots# of BasesSizePublished
SRR19766406,847,715424.6M209.1Mb2016-01-04
SRR19766416,699,363415.4M206.6Mb2016-01-04
SRR19766427,848,384470.9M228.7Mb2016-01-04
SRR19766437,630,004457.8M224.8Mb2016-01-04

ID:
1448455

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