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Sample GSM1480975 Query DataSets for GSM1480975
Status Public on Sep 05, 2014
Title MEF_Mbnl1/2 KO (DKO)-1
Sample type SRA
Source name Mbnl1/2 KO MEFs
Organism Mus musculus
Characteristics strain background: C57J/BL-6 - 129 mix
genotype/variation: Mbnl1/2 KO
tissue: Mouse embryonic Fibroblasts (MEFs)
molecule subtype: oligodT selected total RNA
Treatment protocol MEFs were treated with siRNA against Mbnl3 using Dharmacon 4 transfection reagent for 48 hours in DMEM + 10% FBS
Growth protocol MEFs were grown in DMEM supplemented with 15% FBS
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using Trizol followed by purification with phenol:chloroform extraction. RNA quality was analyzed using Agilent 2100 bioanalyzer
For HITS-CLIP, Quadriceps muscles from WT FVB mice (3 months, n = 3 each genotype) were dissected, frozen in liquid nitrogen, and crosslinked with UV-light (Stratalinker 1800, Stratagene). For MEFs, 10 cm plates (n = 2 each genotype), were UV-crosslinked followed by scraping cells in ice-cold PBS and centrifugation.
For immunoprecipitation, protein lysates were denatured by adding SDS (1% final concentration), heating (100°C/5 min) and subsequent dilution to a final SDS concentration of 0.1% in PXL lysis buffer (Chi et al., 2009) prior to the addition of anti-Mbnl1 (rpAb A2764), Mbnl2 (mAb 3B4) and Mbnl3 (rpAb Mb3/7G) antibodies (5 µg each)
For PolyA-seq, Reverse transcription was carried out using an oligodT containing reverse primer. DNA libraries of 200-500 bp were obtained and sequenced (Illumina) using an oligo dT-containing sequencing primer for 35 bp reads
RNA tags were generated as described (Charizanis et al., 2012) using RNase A (concentrations of 55 U/ml and 0.55 U/ml for high and low RNase, respectively) and cDNA libraries were generated using RNA linkers and primers as described
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer IIx
Description Mbnl1/2 KO MEFs
processed data file: MEF_DKO_pooled.wig.gz
Data processing The raw sequencing reads(FASTQ) were mapped to the reference genome (mm10) using the program OLego (Wu and Zhang, in preparation) with a seed size of 13 nt and allowing ≤ two mismatches or insertions or deletions. Since the antisense strand is sequenced, mapped reads were reverse complemented to obtain the sense strand sequence, and the most 3’ end position was recorded since this position corresponds to the last nucleotide preceding the polyA tail (pA site).
To define robust polyA sites, reads from all samples were pooled and supporting reads for each candidate polyA site were counted. Candidate polyA sites that likely resulted from internal priming from A-rich RNA sequences were then removed using a log-odds model established in a previous study (Derti et al., 2012). These cleaned sites were further filtered to remove those supported by few reads (<10 reads) and then grouped so that sites in each cluster are ≤ 25 nt with each other (Tian et al., 2005)
Finally, unique polyA sites were assigned to overlapping RefSeq and UCSC annotated genes. If a polyA site did not overlap with a known gene, but overlapped with the extended 3' UTR (1 kb extension) of a transcript, it was assigned to that gene. To identify differential polyA site usage, we considered each pair of polyA sites assigned to the same gene using the number of supporting reads in each group of samples.
Statistical analysis was performed using Fisher’s exact test, followed by Benjamini correction (Benjamini and Hochbergh, 1995) for multiple tests and calculation of proportional change of polyA site usage. To minimize multiple test correction, only those relatively abundant polyA sites were considered (coverage≥20, or {site1_WT+site2_WT≥20, site1_KO+site2_KO≥20, site1_WT+site1_KO≥20, site2_WT+site2_KO≥20}).
For hierarchical clustering and principal component analysis, counts per million (CPM) normalized data was log2(n+1) transformed, filtered to exclude low read number (<log25), centered around the mean, and clustered with Cluster 3.0 or subjected to PCA analysis using R. Hierarchical clustering was visualized with Java Tree view and PCA analysis was plotted with the gplot function and rgl library in R.
For HITS-CLIP FASTQ files were filtered to remove low quality reads by requiring a minimum score of 20 in barcode positions and an average score of 20 in the following 25 nucleotides. The filtered reads were then aligned to the mouse reference genome (mm9) by Novoalign. Potential PCR duplicates, as judged from the starting position of genomic mapping and the barcode sequences, were removed to identify unique CLIP tags.
Genome_build: mm9, mm10, hg19
Supplementary_files_format_and_content: Fastq files were aligned to the UCSC genome build (mm9, mm10, or hg19) using BWA, novoaligner (HITS-CLIP) or Olego (polyA-seq). The resulting .sam files were converted to bed files using samtools (HITS-CLIP) or Olego (PolyA-seq). The resulting .bed files were filtered to remove PCR duplicates (HITS-CLIP) and internal A-stretches (polyA-seq). BedGraph files (wiggles) were generated from bedfiles using bedtools (HITS-CLIP) or tag2profile tool from Olego (polyA-seq). Olego is an open source software by Chaolin Zhang lab from Columbia University.
Submission date Aug 18, 2014
Last update date May 15, 2019
Contact name Ranjan Batra
Phone 352-222-9209
Organization name University of California San Diego
Department Cellular and Molecular Medicine
Lab Yeo Lab
Street address 2880 Torrey Pines Scenic Drive
City La Jolla
State/province California
ZIP/Postal code 92037
Country USA
Platform ID GPL11002
Series (1)
GSE60487 Loss of MBNL Leads to Disruption of Developmentally Regulated Alternative Polyadenylation in RNA-Mediated Disease
BioSample SAMN02997315
SRA SRX682634

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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