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Sample GSM1129240

Query DataSets for GSM1129240

Status

Public on May 15, 2013

Title

FFPE_RNaseH

Sample type

SRA

Source name

human normal kidney section (Cybrdi)

Organism

Homo sapiens

Characteristics

total rna input: 1000 ng
rna state: formalin-fixed, paraffin-embedded (FFPE)

Extracted molecule

total RNA

Extraction protocol

Library construction method: RNase H rRNA-depletion (Morlan et al. PLOS One, 2012)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

**********************************************************************

PROCESSED DATA FILE NAME: GSE40705_FFPE_isoform.txt
PROCESSED DATA FILE NAME: GSE40705_FFPE_gene.txt
Removed rRNA reads by aligning reads to rRNA transcripts using BWA version 0.6.1 using default parameters
We created a set of reads that did not map to rRNA to be used for further processing. To do this, reads where neither mate aligned to rRNA were extracted for further use using samtools 0.1.9 using the following commands: samtools view -S -b -f 4 -F 264 rRNA.sam > rRNA.1.bam; samtools view -S -b -f 8 -F 260 rRNA.sam > rRNA.2.bam; samtools view -S -b -f 12 -F 256 rRNA.sam > rRNA.3.bam
Output bam files were merged and reads were extracted using Picard
These reads were aligned to the UCSC transcriptome twice. The first alignment was done to create a set of uniquely mapped reads so that they could be downsampled to a fixed number of reads which are known to map to the transcriptome. This was done using bowtie version 0.12.7 allowing for one hit per read. The following parameters were used: -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 16 -k 1 -m 200
This alignment was then downsampled to a fixed number of reads using Picard
The downsampled portion of the reads were then aligned to the UCSC transcriptome using bowtie version 0.12.7 allowing for multiple hits per read. This was the second, final alignment. The following parameters were used: bowtie -q --phred33-quals -v 2 -e 99999999 -l 25 -I 1 -X 1000 -p 15 -a -m 200
Transcripts and genes were quanitifed using RSEM version 1.1.17 run with default settings using the final Bowtie alignment
Genome_build: UCSC Genome Browser17 knownGene transcript dataset (version 05-Feb-2012)
Supplementary_files_format_and_content: Processed data files are tab-delimited files that consist of the transcripts per million (TPM) expression level values as calculated by RSEM. Results are calculated at the gene (sample_gene.txt) and isoform (sample_isoform.txt) file. Each file contains TPM values calculated for each of the three protocols for the sample (RNaseH, RiboZero, and Total RNA)

Submission date

Apr 24, 2013

Last update date

May 15, 2019

Contact name

Joshua Z Levin

Organization name

Broad Institute of MIT & Harvard

Street address

320 Charles Street

City

Cambridge