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Status |
Public on Sep 10, 2014 |
Title |
HuR fRIP rep2 |
Sample type |
SRA |
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Source name |
K562
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Organism |
Homo sapiens |
Characteristics |
cell line: K562 antibody: HuR
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Treatment protocol |
We collected cells with a gentle 5 minute spin 500g and washed with room temperature PBS. We re-suspended at 5e6 cells per ml in room temperature RPMI media sans FBS or Antibiotic-Antimycotic and added formaldehyde to a final concentration of 0.1%. We cross-linked at room temperature for 10 minutes and then halted it by quenching for 5 minutes at room temperature after adding glycine to a final concentration of 125 mM at a medium pace. We spun cells for 5 minutes 500g and washed 2X in 4°C PBS. We flash froze pellets of 10e6 cells and stored them at -80 °C.
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Growth protocol |
K562 cells (ATCC cat #CCL-243) were grown in RPMI 1640 (Invitrogen, cat #22400105) with 10% FBS and 1% Antiboiotic-Antimycotic 100X (Invitrogen, cat #15240062).
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Extracted molecule |
total RNA |
Extraction 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.
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
CASAVA v1.8.3 used to called bases. Tophat 2.0.9 with default parameters used to align fRIP-Seq reads to hg19 and GENCODE v18 reference annotation. Cuffdiff 2.1.1 used to estimate gene abundances and perform statistical comparisons between the RIP and input alignments. Genome_build: hg19 Supplementary_files_format_and_content: Cuffdiff gene_exp.diff
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Submission date |
Sep 09, 2014 |
Last update date |
May 15, 2019 |
Contact name |
David R Kelley |
E-mail(s) |
dkelley@fas.harvard.edu
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Phone |
732-859-4305
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Organization name |
Harvard University
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Lab |
John Rinn
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Street address |
7 Divinity Ave.
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City |
Cambridge |
State/province |
MA |
ZIP/Postal code |
02138 |
Country |
USA |
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Platform ID |
GPL16791 |
Series (1) |
GSE61238 |
Tranposable elements modulate human mRNAs and lncRNAs via specific RNA-protein interactions. |
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Relations |
SRA |
SRX697723 |
BioSample |
SAMN03031378 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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