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Sample GSM3501186

Query DataSets for GSM3501186

Status

Public on May 02, 2023

Title

Cdk13Δ_c14-1_plusDox_rep2

Sample type

SRA

Source name

Cdk13Δ_c14-1_plusDox

Organism

Mus musculus

Characteristics

strain background: C57Bl/6-129
cell line: V6.5
cell type: Embryonic Stem Cells
genotype: Cdk13-/-; Cdk13 Tg+
clone number: 14-1
dox treatment: +Dox
barcode: GATCAG
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_Library22

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