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Sample GSM3375367 Query DataSets for GSM3375367
Status Public on Sep 05, 2018
Title HeLa_rep5_3N
Sample type SRA
 
Source name HeLa_3N
Organism Homo sapiens
Characteristics tissue: HeLa
protocol: 3'READS+
purpose: 3'READS human samples used in PolyA_DB version 3.2
molecule type: total RNA
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).
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
 
Description 3'READS human samples used in PolyA_DB version 3.2
Data processing Adpter trimming by Cutadapt: cutadapt -a NNNNTGGAATTCTCGGGTGCCAAGG -n 1 -O 10 -m 25 -f fastq -q 10
3'READs and 3'READS+ reads were aligned to hg19 using bowtie2:
For the sample name ends with "3N":
bowtie2 --local --threads 24 -5 3 -x IndexFiles/bowtie2/hg19 -U sample_3N.trimmed.fastq -S sample_3N.sam
For the sample name ends with "4N":
bowtie2 --local --threads 24 -5 4 -x IndexFiles/bowtie2/hg19 -U sample_4N.trimmed.fastq -S sample_4N.sam
For the sample name ends with "6N":
bowtie2 --local --threads 24 -5 6 -x IndexFiles/bowtie2/hg19 -U sample_6N.trimmed.fastq -S sample_6N.sam
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.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include reads count for each PAS position coverved by PolyA_DB version 3.2
 
Submission date Sep 04, 2018
Last update date Sep 06, 2018
Contact name Bin Tian
E-mail(s) btian@rutgers.edu
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 GPL11154
Series (1)
GSE111134 A compendium of conserved cleavage and polyadenylation events in mammalian genes
Relations
BioSample SAMN09976020
SRA SRX4641343

Supplementary file Size Download File type/resource
GSM3375367_HeLa_rep5_3N.readscount.txt.gz 303.7 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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