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Sample GSM2717228 Query DataSets for GSM2717228
Status Public on Jun 13, 2018
Title NT4h_Ft_2_3READS
Sample type SRA
Source name Fibroblast
Organism Mus musculus
Characteristics cell line: NIH3T3
treatment: Untreated, 4 h recovery, with 4sU labeling.
strain: C57BL/6
Treatment protocol Untreated: cells were cultured in normal medium; Sodium Arsenite treatment: 250 µM, 1 h; Recovery: After 1 h sodium arsenite treatment, cells were washed and cultured in normal medium again; 4sU labeling: 1 h (before harvest) culture in medium containing 50 µM of 4-sU.
Extracted molecule total RNA
Extraction protocol TRIzol
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).
RNA-seq: Poly(A)+ RNA in 5 µg of total RNA was purified twice using 10 µl of oligo(dT)25 magnetic beads (NEB) following the manufacturer’s protocol. RNA was then fragmented by partial alkaline hydrolysis (94 °C for 2 min), 3’ dephosphorylated with shrimp alkaline phosphatase, and 5’ phosphorylated with T4 PNK. The RNA fragments were sequentially ligated to a 5’ adenylated 3' blocked 3' adapter (5’-Aden/NNNNNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC, where N is a random nucleotide) with RNA ligase 2 truncated, KQ (200 U/µl) and to a 5′ adaptor (5’-CCUUGGCACCCGAGAAUUCCA) with T4 RNA ligase I. Reverse transcription of ligation product, cDNA amplification by PCR, and library purification were performed as described above for the 3’READS procedure.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
Description Analysis of differential decay of APA isoforms
unlabeled RNA (Flowthrough fraction)
Data processing Adapter trimming performed using Cutadapt
3'READs reads were aligned to mm9 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.
RNAseq reads were aligned to mm9 using STAR
genome build: mm9
Supplementary_files_format_and_content: tab-delimited text files include reads count for each pAs/gene/PoI II position
Submission date Jul 25, 2017
Last update date May 15, 2019
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)
GSE101851 Cellular stress alters 3’UTR landscape through alternative polyadenylation and isoform-specific degradation
BioSample SAMN07414004
SRA SRX3033673

Supplementary file Size Download File type/resource
GSM2717228_NT4h_Ft_2.readscount.txt.gz 153.6 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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