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Sample GSM1659706

Query DataSets for GSM1659706

Status

Public on Jan 01, 2016

Title

adar_rep2_fRIP-seq

Sample type

SRA

Source name

K562 cells

Organism

Homo sapiens

Characteristics

frip antibody: Sigma,HPA003890,HPA003890
passage: four to seven
cell line: K562

Treatment protocol

CROSSLINKING: 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

Growth protocol

K562 cells (ATCC cat #CCL-243) were grown in RPMI 1640 (Invitrogen, cat #22400105) with 10% FBS and 1% Antibiotic-Antimycotic 100X (Invitrogen, cat #15240062).

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.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

fRIP-seq/RNA-seq

Data processing

Basecalls performed using CASAVA version 1.4
We aligned sequencing reads to human genome assembly hg19 and GENCODE v18 reference annotation (Harrow et al. 2012) using TopHat (Trapnell, Pachter, and Salzberg 2009) using the -M option
We estimated transcript and gene abundances per REPLICATE as well as depletion/ enrichment calls using the Cuffdiff 2 cuffNorm option
We made significance calls to date matched input samples per CONDITION using Cuffdiff 2 with the -u option
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample at gene and isoform level in addition to CuffDiff 2 generated p and q values with significance calls

Submission date

Apr 16, 2015

Last update date

May 15, 2019

Contact name

David R Kelley

E-mail(s)

dkelley@fas.harvard.edu

Phone

732-859-4305

Organization name

Harvard University