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Sample GSM2595502

Query DataSets for GSM2595502

Status

Public on May 01, 2018

Title

MC4R-/- rep3

Sample type

SRA

Source name

Left ventricle posterior wall

Organism

Mus musculus

Characteristics

strain: C57BL6/J
tissue: Left ventricle posterior wall
age: 30 weeks

Growth protocol

Mice were generated using a het-het breeding strategy and housed at 24degC for 30 weeks.

Extracted molecule

polyA RNA

Extraction protocol

Mice were anethetized with TBE and then hearts were flash frozen in liquid nitogen. Total RNA was extracted usign trizol with a quiagen column cleanup with on column DNAse.
200ng of DNase-treated total RNA with a RNA integrity number greater than 7 was used to generate polyA-enriched mRNA libraries using KAPA Stranded mRNA sample kits with indexed adaptors (Roche; Basel, Switzerland).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

3472-MJL-4_1

Data processing

Illumina HiSeq300 reads were converted to FASTQ files using the Bcl2fastq2 conversion software.
Read quality was checked using FastQCv0.11.5. TopHat v2.1.0 was then used for read allignment Tool Parameters Is this single-end or paired-end data? paired_collection RNA-Seq FASTQ paired reads No dataset collection. Mean Inner Distance between Mate Pairs 300 Std. Dev for Distance between Mate Pairs 20 Report discordant pair alignments? Yes Use a built in reference genome or own from your history indexed Select a reference genome mm10 TopHat settings to use full Max realign edit distance 1000 Max edit distance 2 Library Type FR First Strand Final read mismatches 2 Use bowtie -n mode No Anchor length (at least 3) 8 Maximum number of mismatches that can appear in the anchor region of spliced alignment 0 The minimum intron length 70 The maximum intron length 500000 Allow indel search Yes Max insertion length. 3 Max deletion length. 3 Maximum number of alignments to be allowed 20 Minimum intron length that may be found during split-segment (default) search 50 Maximum intron length that may be found during split-segment (default) search 500000 Number of mismatches allowed in each segment alignment for reads mapped independently 2 Minimum length of read segments 25 Output unmapped reads False Do you want to supply your own junction data Yes Use Gene Annotation Model Yes Gene Model Annotations 220: UCSC Main on Mouse: refGene (genome) Use Raw Junctions No Only look for supplied junctions No Use coverage-based search for junctions auto Use Microexon Search No Do Fusion Search No Set Bowtie2 settings No Specify read group? no Job Resource Parameters no
Aligned reads were then assembled into transcripts with Cufflinks v2.2.1 Tool Parameters: SAM or BAM file of aligned RNA-Seq reads 240: TopHat on data 220, data 33, and data 22: accepted_hits Max Intron Length 300000 Min Isoform Fraction 0.1 Pre MRNA Fraction 0.15 Use Reference Annotation Use reference annotation Reference Annotation 220: UCSC Main on Mouse: refGene (genome) Count hits compatible with reference RNAs only No Perform Bias Correction Yes Reference sequence data cached Using reference genome mm10 Use multi-read correct No Apply length correction Cufflinks Effective Length Correction Global model (for use in Trackster) No dataset. Set advanced Cufflinks options Yes Library prep used for input reads fr-firststrand Mask File Inner mean distance 300 Inner distance standard deviation 20 Max MLE iterations 5000 Alpha value for the binomial test used during false positive spliced alignment filtration 0.001 percent read overhang taken as suspiciously small 0.09 Intronic overhang tolerance 8 Maximum genomic length of a given bundle 3500000 Maximum number of fragments per locus 1000000 Minimal allowed intron size 50 Minimum average coverage required to attempt 3prime trimming. 10 The fraction of average coverage below which to trim the 3prime end of an assembled transcript. 0.1 Job Resource Parameters no
Files were then merged using Cuffmerge v2.2.1 and RMPK values were quantified using CuffQuant v2.2.1:Tool Parameters Transcripts 341: Cuffmerge on data 220, data 333, and others: merged transcripts Add replicate 225: TopHat on data 220, data 39, and data 38: accepted_hits Use multi-read correct False Perform Bias Correction Yes Reference sequence data cached Using reference genome mm10 apply length correction cufflinks effective length correction Set Additional Parameters for single end reads? (not recommended for paired-end reads) No Set Advanced Cuffquant parameters? Yes Library prep used for input reads fr-firststrand Mask File Max MLE iterations 5000 Maximum number of fragments per locus 500000 Job Resource Parameters no
Cuffnorm v2.2.1.1 was then used to normalize for transcript length: Tool Parameters Transcripts 341: Cuffmerge on data 220, data 333, and others: merged transcripts Input data type CXB Name WT Replicates 442: Cuffquant on data 225 and data 341: Abundances.cxb , 443: Cuffquant on data 230 and data 341: Abundances.cxb , 444: Cuffquant on data 235 and data 341: Abundances.cxb , 445: Cuffquant on data 240 and data 341: Abundances.cxb Name KO Replicates 447: Cuffquant on data 250 and data 341: Abundances.cxb , 448: Cuffquant on data 255 and data 341: Abundances.cxb , 449: Cuffquant on data 260 and data 341: Abundances.cxb , 450: Cuffquant on data 265 and data 341: Abundances.cxb , 451: Cuffquant on data 270 and data 341: Abundances.cxb , 452: Cuffquant on data 275 and data 341: Abundances.cxb Library normalization method geometric Include Read_Group/Attribute Datasets Yes output_format Simple Table Set Advanced Cuffnorm parameters? Yes Library prep used for input reads fr-firststrand Hits included in normalization Compatible Hits Job Resource Parameters no
Differnetial gene trasncription testing was then conducted using Cuffdiff v2.2.1.3: Tool Parameters Library normalization method geometric Dispersion estimation method pooled False Discovery Rate 0.05 Min Alignment Count 10 Use multi-read correct False Perform Bias Correction No Include Read Group Datasets No Include Count Based output files No apply length correction cufflinks effective length correction Set Additional Parameters for single end reads? (not recommended for paired-end reads) No Set Advanced Cuffdiff parameters? No Job Resource Parameters no
Genome_build: mm10
Supplementary_files_format_and_content: cuffnorm_allsamples.txt: Tab-delimited text file with a matrix of all RPKM values. Cuffdiff_q_0.05.txt: Tab-delimited text file of log fold change RPKM values with q<0.05

Submission date

May 02, 2017

Last update date

May 15, 2019

Contact name

Michael Litt

E-mail(s)

michael.j.litt@Vanderbilt.Edu

Organization name

Vanderbilt University

Department

MPB