GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3030277

Query DataSets for GSM3030277

Status

Public on May 20, 2018

Title

GoldCLIP_YFP_UVC_replicate1

Sample type

SRA

Source name

Human Embryonic Kidney 293 cells

Organism

Homo sapiens

Characteristics

cell line: 293T
genotype: Halo-YFP transgenetic

Growth protocol

Transgenetic 293 cells were routinely generated by re-plating with DMEM media supplimently with 10% FBS (typically 2–3 × 10^6 cells into a T75 flask). Puromycin was added to a final concentration of 0.5 μg/ml.

Extracted molecule

total RNA

Extraction protocol

Lysates were lysed in PBS supplemented with 1% Triton, clarified from debri, and HaloTag-protein-RNA complexes were isolated using Magne Halo Beads (Promega).
Libraries were prepared according to the iCLIP library construction protocol (Konig, Zarnack et al. 2011). Briefly, RNA in the Halo-RBP-RNA complex was released and reverse transcribed. Then cDNAs were gel purified and cyclized. The cyclized cDNAs were annealed to a complementary oligo which allows BamHI digestion to linearize the cDNAs The linearized cDNAs were then amplified by PCR to add Illumina adaptors. The final libraries were purified and sequenced on either Illumina X10 or HiSeq 2500 platform.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

HiSeq X Ten

Description

GoldCLIP
GoldCLIP_YFP_UVC.norm.plus.bw
GoldCLIP_YFP_UVC.norm.minus.bw

Data processing

Sequenced reads were trimmed for adaptor sequence and masked for low-quality sequence. If multiple reads have exactly the same insert sequence and random barcode, only one is kept, after that, the random barcode and experimental barcode (positions 1-9) were removed.
The processed reads were first mapped to the spike-in genome (dm3, download from UCSC) using Bowtie v1.1.2 with parameters -f -p 8 -v 2 -k 1--best --sam --un, unmapped reads in previous step were mapped to a library of mature rRNAs, tRNAs and the mitochondrial genome by allowing multiple hits (rRNAs: RefSeq id, NR_023363.1, NR_003285.2, NR_003287.2, and NR_003286.2; tRNAs: downloaded from Genomic tRNA Database (http://gtrnadb.ucsc.edu) and only those from Homo_sapiens were retained, and CCA was appended to the 3′ end of the sequences). Mapping was performed using Bowtie v1.1.2 with parameters -f -p 8 -v 2 -k 1--best --sam --un. The remaining unmapped reads were aligned to the human genome (hg19) using Bowtie with parameters -f -p 8 -v 2 -m 1 --best --strata --sam.
Peak identification was performed on uniquely mapped reads by CLIPper4 (available at https://github.com/YeoLab/clipper/releases/tag/1.0) was done with options -s hg19 -o --bonferroni --superlocal --threshold-method binomial --save-pickle.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files containing RT stops of GoldCLIP datasets mapped to the hg19 build of the human genome. Add data are normalized to a library depth of 10,000,000 reads. Bed files containing peaks of GoldCLIP datasets identified by CLIPper.

Submission date

Mar 05, 2018

Last update date

May 20, 2018

Contact name

Ming Wang

E-mail(s)

wangming@ibp.ac.cn

Phone

01064881022

Organization name

Institute of Biophysics, Chinese Academy of Sciences