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Sample GSM2221314 Query DataSets for GSM2221314
Status Public on Oct 28, 2016
Title PA1_S200D_IP_rep1
Sample type SRA
 
Source name PA1_S200D
Organism Homo sapiens
Characteristics cell line: PA1
cell type: Embryonal carcinoma cells
transgene: FLAG-LIN28 (S200D)
rip antibody: FLAG M2 (Sigma #A2220)
Growth protocol PA1 cells (ATCC# CRL-1572) 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 PA1 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 NextSeq 500
 
Description polyA+ RNA
RIP1_PA1_RPKM.xls
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 May 15, 2019
Contact name Areum Han
E-mail(s) areum.han@childrens.harvard.edu
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 GPL18573
Series (2)
GSE83905 RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT) or phospho-mimetic (S200D) FLAG-LIN28A in PA1 cells
GSE83906 RNA-seq analysis of mRNAs immunoprecipitated by wild-type (WT), phospho-mimetic (S200D), or phospho-null (S200A) FLAG-LIN28A
Relations
BioSample SAMN05331285
SRA SRX1888933

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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