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Sample GSM1865365 Query DataSets for GSM1865365
Status Public on Jan 15, 2016
Title siSki8_3READs_2
Sample type SRA
Source name siSki8 for 3'READS, batch 2
Organism Mus musculus
Characteristics cell line: C2C12
cell condition: myoblasts
library protocol: 3'READS
Treatment protocol For transient transfection of siRNA, cells were seeded at 104 cells cm−2 for 48 h transfection. 5 μl RNAiMax in 2 ml medium and 50 nM siRNA were applied to cells by using fast-forward method.
Growth protocol C2C12 myoblasts (Sigma) were grown and induced to differentiate into myotubes as described [Blais, A., et al. 2007].
Extracted molecule total RNA
Extraction protocol Preparation of solubilized chromatin fraction and immunoprecipitation: Cell pellets were resuspended in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol (DTT), and protease inhibitors (aprotinin, leupeptin, pepstatin A, and phenylmethyl sulphonyl fluoride)), supplemented with Triton X-100 (0.1%), and incubated on ice for 5 minutes. The nuclear pellet was separated from the cytoplasmic fraction, washed once with buffer A, and collected by centrifugation. Nuclei were lysed with buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM dithiothreitol (DTT), and protease inhibitors (aprotinin, leupeptin, pepstatin A, and phenylmethyl sulphonyl fluoride)). The solubilized chromatin fraction was separated from nucleoplasm by centrifugation at 2250 g for 4 minutes at 4°C, washed once with buffer B, and collected by centrifugation. For immunoprecipitations, the chromatin pellet was resuspended with binding buffer (20 mM Hepes pH7.9, 100 mM potassium chloride, 0.2 mM EDTA, 20% glycerol, 0.5 mM dithiothreitol (DTT), and 0.5mM AEBSF). 1 mg of protein was immunoprecipated, and antibody-protein A sepharose beads were washed three times with buffer (50 mM Hepes pH7.9, 250 mM sodium chloride, 5 mM EDTA, 0.50% NP-40, and 10% glycerol) prior to SDS–PAGE and immuno-blotting.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
Data processing RNA-seq data analysis: Reads were mapped to the mouse genome (mm9) using bowtie2 (local mode). Uniquely mapped reads with MAPQ score>10 were used. Refseq gene model was used to calculate gene expression level. For protein-coding genes, only reads mapped to coding region were used to eliminate the effect of 3’UTR alternative polyadenylation in gene expression calculation. Reads per kilobase per million total uniquely mapped reads (RPKM) value was calculated to reflect gene expression level. Significantly regulated genes were selected based on these criteria: >1.4-fold difference of RPKM between Paf1C siRNA treated cells and siRNA control cells and P value<0.01 (Fisher’s exact test).
3’READS and data analysis. 3’READS reads were mapped to the mouse genome (mm9) using bowtie2 (local mode). Uniquely mapped reads (with MAPQ score > 10) that had at least two additional 5’ Ts not aligned to genome (presumably derived from the poly(A) tail) were named pA site supporting (PASS) reads and were used to calculate pA isoform expression. pA isoforms expressed below 5% of all isoforms of a gene were discarded. Significantly regulated APA events were identified by the Significance Analysis of Alternative Polyadenylation (SAAP) method [Li et al., 2015].
ChIP-seq data analysis: Reads were mapped to the mouse genome (mm9) using bowtie2. Reads non-uniquely mapped (MAPQ≤10) were discarded. Multiple reads mapped to same genomic loci (defined by chromosome, strand, start and end position) were collapsed to one. To calculate enrichment score of a genomic locus in a meta-gene plot, the first nt at the 5’end of a read was counted for all genes in a group and converted to read per million total reads (RPM) value. The enrichment score was defined by log2 ratio of RPM between IP sample and input sample. The relative genomic location was binned (e.g., every 50 nt relative to TSS or every percent in gene body) before calculating enrichment score and then plotted in R.
Genome_build: mm9
Supplementary_files_format_and_content: contains information of gene symbol, pA coordinates, read number of pAs in different 3'READS samples. "pAtype" is pA type based on location of a pA (I, intron; E, upstream exons; 3, pAs in 3'most exon; UA, pA in upstream antisense of a protein-coding gene.). "" contains information of gene symbol, Refseq transcript name, read number and RPKM (read per kb per million total reads) values. Bigwig files: for visualization of ChIP-seq data, all uniquely mapped and collapsed reads were used. Reads were extended from its 5’end mapped position by 250 nts (reflecting the average fragment size in ChIP-seq DNA libraries) and converted to bigwig format files using an in-house perl script and bedGraphToBigWig program from UCSC genome browser.
Submission date Sep 01, 2015
Last update date May 15, 2019
Contact name Wencheng Li
Organization name PTC Therapeutics
Street address 100 Corporate Count
City South Plainfield
State/province NJ - New Jersey
ZIP/Postal code 07080
Country USA
Platform ID GPL13112
Series (1)
GSE72574 Paf1C plays novel subunit-specific roles in alternative cleavage and polyadenylation
BioSample SAMN04025277
SRA SRX1176712

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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