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Status |
Public on Mar 31, 2015 |
Title |
S3 proximal tubule, replicate #4 |
Sample type |
SRA |
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Source name |
Microdissected renal tubule segment
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Organism |
Rattus norvegicus |
Characteristics |
age: Adult Sex: male strain: Sprague-Dawley tissue: S3 proximal tubule
|
Treatment protocol |
The left kidney of a male Sprague-Dawley rat was isolated from the circulation and perfused with 1x dissection solution and collagenase solution. The chunks of the kidney were digested in the same collagenase solution at 37 C for 25 minutes (cortex), 50 minutes (outer medulla), or 90 minutes (inner medulla).
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Growth protocol |
Male Sprague-Dawley rats were grown and maintained in the Animal Facility of the NHLBI. Normal rat chow was fed to the animals. Rats weighing 200-250 g were used for this study.
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Extracted molecule |
total RNA |
Extraction protocol |
Dissected tubules were lysed in buffer containing RNase inhibitors and universal primers. Reverse transcription was started in this cell lysate. See Tang F. et al. (2010). "RNA-Seq analysis to capture the transcriptome landscape of a single cell" Nature Protocol 5(3):517-35. We followed an RNA-seq protocol developed for single-cell RNA-seq. See Tang F. et al. (2010). "RNA-Seq analysis to capture the transcriptome landscape of a single cell" Nature Protocol 5(3):517-35. cDNAs were sheared and made into adapter-ligated RNA-seq libraries using NuGen Mondrian machine and Ultralow Library System. We sequenced from both ends of a cDNA molecule, 50 nucleotides each.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
mRNA released from cell lysate S3_4
|
Data processing |
Base-calling was performed using on-instrument real-time analysis (RTA) pipeline provided by Illumina HiSeq 2000. Individual pair of FASTQ sequences was inspected and nucleotides with phred33 score lower than 30 were trimmed with Trimmomatic 0.32 (http://www.usadellab.org/cms/?page=trimmomatic) using the following command: java -jar trimmomatic-0.32.jar PE -threads 8 -phred33 sample_1.fastq.gz sample_2.fastq.gz sample1_p1.fastq. sample1_up1.fastq sample2_p2.fastq sample2_pup2.fastq TRAILING:30 MINLEN:35 Pairs of FASTQ sequences that survived the trimming process were mapped to rat reference genome (rn5) with STAR 2.3.0-r192 using the following command: STAR --genomeDir <genome directory> --readFilesIn sample_p1.fastq sample_p2.fastq --runThread 8 --outFilterMismatchNmax 3 --genomeLoad LoadAndKeep --outSamstrandField intronMotif --alignIntronMax 10000 --alignMatesGapMax 10000 --outFilterIntronMotifs RemoveNoncanonicalUnannotated The raw mapping data was filtered for uniquely mapped reads using SAMtools. HOMER software package was used to calculate RPKMs. PA-seq data were mapped to Rn5 using Burrows-Wheeler Aligner. Peaks were called using in-house software written in Python. See Ni, T., Y. Yang, et al. (2013). "Distinct polyadenylation landscapes of diverse human tissues revealed by a modified PA-seq strategy." BMC Genomics 14:615. Genome_build: RGSC 5.0/rn5 Supplementary_files_format_and_content: RPKM.without.PAseq.txt is a tab-delimited text file that contains a matrix of reads per kilobase exons per million mapped reads (RPKMs) for RefSeq transcripts without information from PA-seq study. RPKM.with.PAseq.txt is a tab-delimtied text file that contains a matrix of revised RPKMs after including the information on alternative polyadenylation from PA-seq study. Bedgraph files for individual tubule segments, in bedgraph format, contain median read counts at each nucleotide normalized to million uniquely mapped reads. These files can be uploaded onto the UCSC genome browser for visualization. PAseq.peak.rn5 bedgraph files for the forward and reverse strand contain information on polyadenylation peaks in the whole kidney across the rat genome. These files can be uploaded onto the UCSC genome browser to examine the location and intensity of polyadenylation peaks.
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Submission date |
Apr 11, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Jae Wook Lee |
E-mail(s) |
leejw4@mail.nih.gov
|
Organization name |
National Institutes of Health
|
Street address |
10 Center Drive, 6N314
|
City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
|
|
Platform ID |
GPL14844 |
Series (1) |
GSE56743 |
RNA-seq profiling of microdissected rat renal tubule segments |
|
Relations |
BioSample |
SAMN02726170 |
SRA |
SRX516053 |