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Sample GSM3022828 Query DataSets for GSM3022828
Status Public on Aug 08, 2018
Title FVB testis replicate 2
Sample type SRA
Source name testis
Organism Mus musculus
Characteristics tissue: testis
protocol: 3'READS
purpose: Analysis of APA in heart, testis and heart between mouse and rat
strain: FVB/NJ
Treatment protocol NA
Extracted molecule total RNA
Extraction protocol TRIzol
See the "extraction protocol" column in the SAMPLES section.
3'READS: The 3’READS procedure has been described in (Zheng et al., 2016). Briefly, Poly(A)+ RNA in 0.1-15 µg of total RNA was captured using 10 µl of oligo(dT)25 magnetic beads in 100 µl 1x binding buffer (10 mM Tris-Cl, pH7.5, 150 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20) and fragmented on the beads using 1.5 U of RNase III in 30 µl RNase III buffer (10 mM Tris-Cl pH8.3, 60 mM NaCl, 10 mM MgCl2, and 1 mM DTT) at 37 °C for 15 min. After washing away unbound RNA fragments with binding buffer, poly(A)+ fragments were eluted from the beads with TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5) and precipitated with ethanol, followed by ligation to 3 pmol of heat-denatured 5’ adapter (5’-CCUUGGCACCCGAGAAUUCCANNNN) in the presence of 1 mM ATP, 0.1 µl of SuperaseIn, and 0.25 µl of T4 RNA ligase 1 in a 5 µl reaction at 22 °C for 1 h. The ligation products were captured by 10 pmol of biotin-T15-(+TT)5 attached to 12 µl of Dynabeads MyOne Streptavidin C1. After washing with washing buffer (10 mM Tris-Cl pH7.5, 1 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20), RNA fragments on the beads were incubated with 0.01 U/µl of RNase H at 37 °C for 30 min in 30 µl of RNase H buffer (50 mM Tris-Cl pH 7.5, 5 mM NaCl, 10 mM MgCl2, and 10 mM DTT). After washing with RNase H buffer, RNA fragments were eluted from the beads in elution buffer (1 mM NaCl, 1 mM EDTA, and 0.05% TWEEN 20) at 50 °C, precipitated with ethanol, and then ligated to 3 pmol of heat-denatured 5’ adenylated 3’ adapter (5’-rApp/NNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC) with 0.25 µl T4 RNA ligase 2 (truncated KQ version) at 22 °C for 1 h in a 5 µl reaction containing 15% PEG 8000 and 0.2 µl of SuperaseIn. The ligation products were then precipitated and reverse transcribed using M-MLV reverse transcriptase, followed by PCR amplification using Phusion high-fidelity DNA polymerase and bar-coded PCR primers for 13-18 cycles (13 cycles for 5 µg input, 15 cycles for 1 µg input, and 18 cycles for inputs below 1 µg). PCR products were size-selected twice with AMPure XP beads, using 0.6 volumes of beads (relative to the PCR reaction volume) to remove large DNA molecules and an additional 0.4 volumes of beads to remove small DNA molecules. The eluted DNA was selected again with 1 volume of AMPure XP beads to further remove small DNA molecules. The size and quantity of the libraries eluted from the AMPure beads were examined using a high sensitivity DNA kit on an Agilent Bioanalyzer. Libraries were sequenced on an Illumina NextSeq 500 (1x75 bases).
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
Data processing Adpter trimming performed using Cutadapt
3'READs reads were aligned to mm9/rn5 using bowtie2
For 3'READs data, reads with a mapping quality score (MAPQ) ?10 were kept for further analysis. Reads with ?2 non-genomic 5’Ts after alignment were called polyA site supporting (PASS) reads. pAs within 24 nt of each other were clustered. pAs with less than 5 PASS reads in all the samples were first trimmed.
Genome_build: mm9 for mouse, rn5 for rat
Supplementary_files_format_and_content: tab-delimited text files include reads count for each PAS position
Submission date Feb 26, 2018
Last update date Aug 08, 2018
Contact name Bin Tian
Organization name Rutgers New Jersey Medical School
Department Department of Microbiology, Biochemistry and Molecular Genetics
Street address 205 South Orange Ave.
City Newark
State/province NJ
ZIP/Postal code 07103
Country USA
Platform ID GPL19057
Series (1)
GSE111134 A compendium of conserved cleavage and polyadenylation events in mammalian genes
BioSample SAMN08608926
SRA SRX3742708

Supplementary file Size Download File type/resource
GSM3022828_FVB_testis_replicate_2.readscount.txt.gz 713.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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