|
| Status |
Public on Jun 10, 2017 |
| Title |
Day2_WaterLoaded1 |
| Sample type |
SRA |
| |
|
| Source name |
Microdissected cortical collecting duct
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
Sex: male
developmental stage: adult
tissue: cortical collecting duct
|
| Treatment protocol |
For vasopressin-escape experiment, subcutaneous minipumps containing DDAVP (5ng/hr) were implanted into the back of the rats. These rats received either water load ("WL", 50 mL/day) or normal amount of water ("C", 25 mL/day) in the form of gelled food. After 4 days of water load, the cortical collecting duct was microdissected.
|
| 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. For vasopressin-escape experiment, 5-week-old rats were used.
|
| 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.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Cortical collecting duct, Water-loaded rat #1, Day 2
|
| 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 (ensembl Rnor6.0) with STAR 2.4.0.1 using the following command: STAR --genomeDir <genome directory> --readFilesIn sample_p1.fastq sample_p2.fastq --runThread 8 --outFilterMismatchNmax 3 --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outFilterMismatchMax 3 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonicalUnannotated
The raw mapping data was filtered for uniquely mapped reads using SAMtools.
HTSeq-count and R were used to count mapped reads and calculate FPKMs.
Genome_build: Ensembl Rnor6.0
Supplementary_files_format_and_content: vasopressin_escale_rn6_FPKM.txt is a tab-delimited text file that contains a matrix of fragments per kilobase exons per million mapped reads (RPKMs) for Ensembl Rnor6.0 genes. Vasopressin_escape_rn6_htseqcount.txt is a tab-delimited text file that contains a matrix of read counts for Ensemble Rnor6.0 genes.
|
| |
|
| Submission date |
Jun 11, 2015 |
| 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) |
| GSE69779 |
RNA-seq profiling of cortical collecting ducts in rats undergoing vasopressin escape |
|
| Relations |
| BioSample |
SAMN03770004 |
| SRA |
SRX1056369 |
