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Sample GSM3501188 Query DataSets for GSM3501188
Status Public on May 02, 2023
Title Cdk13Δ_c11-8_minusDox48_rep2
Sample type SRA
 
Source name Cdk13Δ_c11-8_minusDox48
Organism Mus musculus
Characteristics strain background: C57Bl/6-129
cell line: V6.5
cell type: Embryonic Stem Cells
genotype: Cdk13-/-; Cdk13 Tg+
clone number: 11-8
dox treatment: -Dox 48 hours
barcode: GGCTAC
rin value: 8.9
Treatment protocol Cdk13∆ clones were maintained in 1ug/mL doxycycline (dox) (Sigma) in ES media (changed daily) to sustain complementing levels of Cdk13. To investigate Cdk13 loss, cells were washed at time zero with HBS and switched to ES media without dox for 48 or 72 hours.
Growth protocol V6.5 (C57Bl/6-129) mESCs and derived cell lines were cultured on 0.2% gelatin-coated tissue culture plates in ES media (HEPES-buffered DMEM (Thermo Fisher) supplemented with 15% FBS (Hyclone), 1000U/mL LIF (Millipore), non-essential amino acids, L-glutamine, BME, penicillin, and streptomycin). Cdk13∆ clones were maintained in 1ug/mL doxycycline (dox) (Sigma) in ES media (changed daily) to sustain complementing levels of Cdk13. To investigate Cdk13 loss, cells were washed at time zero with HBS and switched to ES media without dox for 48 or 72 hours.
Extracted molecule polyA RNA
Extraction protocol RNA Sequencing: Total RNA was harvested using Trizol Reagent (Thermo Fischer) following the manufacturer's protocol and subsequently DNase treated with Turbo DNase (Thermo Fishcer) under standard reaction conditions. RNA quality was assessed by the Agilent 2100 BioAnalyzer and only samples with a RIN value >= 8.9 were used for library preparation.
RNA Sequencing: Libraries were made from 1ug of total RNA input using the TruSeq Stranded mRNA Library Prep Kit (Illumina RS-122-2102) with multiplexing barcodes, following the standard protocol with the following specifications: (1) 5 min RNA fragmentation time, (2) Superscript III (Thermo Fisher) was used for reverse transcription, (3) 15 cycles of PCR were used during the library amplification step, and (4) AMPure beads (Beckman Coulter) were used to size select/purify the library post PCR amplification instead of gel size selection.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Description 150927_C5FHE_1572A_L1_1_Library24
Data processing Raw RNA-seq reads were mapped using STAR aligner version 2.4.1d with the following parameters (Note: Boutz_Mm9_Master_05_06_13_mod_base1_1.juncs is a custom annotated master junctions file for mouse embryonic stem cells and can be made available upon request.): STAR --runMode alignReads --runThreadN 8 --genomeDir UCSC_mm9 --sjdbFileChrStartEnd Boutz_Mm9_Master_05_06_13_mod_base1_1.juncs --twopassMode Basic --sjdbOverhang 74 --outReadsUnmapped Fastx --outSAMtype BAM Unsorted SortedByCoordinate --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 70 --alignIntronMax 500000 --alignMatesGapMax 500000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outSAMstrandField intronMotif --outFilterType BySJout
We used custom Python scripts to derive a transcriptome annotation based on Mm9 (Mus_musculus_NCBI_build37.1) gene start and end boundaries, the location of polyadenylation sites as determined by 3’end sequencing (Almada et al, 2013), and the genomic locations of all mapped splice junctions from the RNA-seq data. Mapped splice junctions detected in all samples were combined and processed using custom Python scripts to filter out junctions representing < ~1% of transcripts. Alternative and constitutive intron classifications were performed using custom Python scripts, and are agnostic with regard to existing annotations other than known gene boundaries. If no overlapping introns exist for a given intron, it is assigned to the constitutive class. The subgroups containing overlapping introns are assigned a splicing classification if the start and end coordinates of all of the constituent introns fall into a pattern representing a known splice type (cassette, mutually exclusive, alternative 5' splice site, alternative 3' splice site). Annotation of the distal polyadenylation site isoform for each gene was based on the consensus isoform, i.e. the junctions defining each exon are the most frequently used junction detected among all of the combined samples. Each IPA site within a gene was then assigned to an additional transcript based on the consensus isoform but terminating at the IPA cleavage site. These gene annotations were then converted to DEXseq exon parts using DEXseq-associated script dexseq_prepare_annotation.py and the reads mapping to each exon part in each sample were counted using dexseq_count.py. Using the counts matrix thus derived for all biological replicates in each condition, DEXseq was used to identify changes in the relative abundance of each exon part as normalized to all exon parts within the gene, including those representing alternative splicing of internal introns as well as the IPAs and distal polyadenylation site isoform 3’ terminal exon. This gave a log2-fold change and FDR adjusted p value for each exon part as it differed between the Cdk13-expressing and Cdk13-depleted samples. IPA sites or distal polyadenylation site isoform 3’ terminal exons whose exon part exhibited an adjusted p value < 0.05 were considered statistically significant. Significantly changing alternative exons,IPA sites and distal polyadenylation site isoform 3’ terminal exons are listed in the Cdk13_48_72_All_DEXSeq_results.xslx processed data file.
Genome_build: mm9
Supplementary_files_format_and_content: Excel table containing annotated exon parts, quantification and determination of statistical significance for changes in exon part expression between Cdk13-expressing and -depleted cells.
 
Submission date Dec 04, 2018
Last update date May 02, 2023
Contact name Sara Jane Dubbury
E-mail(s) dubbury@mit.edu
Phone 425-501-0761
Organization name MIT
Department Koch Institute
Lab Phillip Sharp
Street address 500 Main Street, 76-417
City Cambridge
State/province MA
ZIP/Postal code 02139
Country USA
 
Platform ID GPL19057
Series (2)
GSE123339 Oncogenic CDK13 Mutations Impede Nuclear RNA Surveillance (mouseRNAseq)
GSE131334 Oncogenic CDK13 Mutations Impede Nuclear RNA Surveillance
Relations
BioSample SAMN10520184
SRA SRX5095472

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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