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Sample GSM2221308 Query DataSets for GSM2221308
Status Public on Oct 28, 2016
Title HeLa_WT_IP_rep3
Sample type SRA
Source name HeLa_WT_IP
Organism Homo sapiens
Characteristics cell line: HeLa
cell type: Cervical carcinoma cells
transgene: FLAG-LIN28 (WT)
rip antibody: FLAG M2 (Sigma #A2220)
Growth protocol HeLa cells were maintained in DMEM/10% FBS.
Extracted molecule polyA RNA
Extraction protocol Cells were harvested in cold PBS and immediately lysed in M2 lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing 100 U/ml RNasin (Promega) and 2X protease and phosphatase inhibitors (Pierce). FLAG-tagged LIN28 variants were purified using the anti-FLAG M2 affinity gel following the manufacturer’s specifications (Sigma). Parental HeLa cells (no FLAG) were used as controls for antibody specificity. RNA was isolated from the beads using Trizol (Invitrogen) and RNeasy columns (Qiagen). Purified RNA was subjected to polyA selection using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB).
Libraries were prepared with the NEBNext Ultra RNA Library Prep Kit (NEB).
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2500
Data processing From the raw reads, we removed adaptor sequences using a Cutadapt tool, allowing some mismatches (The specific parameters we used were -n 2 -e 0.1 -O 15 --quality-base=33).
Then, the cleaned reads were aligned to the human genome/transcriptome (hg19 and corresponding UCSC gene model) using a TopHat2 software with the following parameters (--library-type=fr-firststrand --min-intron-length=10 --no-coverage-search --microexon-search --min-isoform-fraction=0).
To estimate expression levels for each gene, we counted aligned reads per gene and using a htseq-count tool.
And calculated CPM (Counts Per Million) values were converted to RPKM (Reads Per Kilobase per Million mapped reads) values corresponding to gene lengths.
Genome_build: hg19
Supplementary_files_format_and_content: excel, RPKM values of genes after corresponding immunoprecipitation
Submission date Jun 30, 2016
Last update date Oct 28, 2016
Contact name Areum Han
Organization name Boston Children's Hospital
Lab Daley lab
Street address Karp Family Building, 7th Floor. 300 Longwood Ave.
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
Platform ID GPL16791
Series (2)
GSE83904 RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT) or phospho-null (S200A) FLAG-LIN28A in HeLa cells
GSE83906 RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT), phospho-mimetic (S200D), or phospho-null (S200A) FLAG-LIN28A
BioSample SAMN05331279

Supplementary data files not provided
Raw data provided as supplementary file
Processed data are available on Series record

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