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Sample GSM6523086 Query DataSets for GSM6523086
Status Public on Jun 01, 2023
Title MelanA cells poly(A) seq E
Sample type SRA
 
Source name Melan-A
Organism Mus musculus
Characteristics cell line: Melan-A
cell type: immortalized melanocyte
genotype: WT
treatment: none
Extracted molecule polyA RNA
Extraction protocol RNA was harvested using Direct-zol RNA MiniPrep kit (Zymo Research)
Poly(A)-seq:.RNA was isolated from cell pellets with the Direct-zol RNA MiniPrep kit (Zymo Research). Protocol was adapted from Derti et al. 2012. In brief, 5 ug of RNA was heated at 65 degrees for 2 minutes and then placed on ice. Poly(A) selected mRNA was isolated using DynaBeads (ThermoFischer 61006) per the manufacturer’s instructions. 1st strand cDNA synthesis using SuperScript IV Reverse Transcriptase (ThermoFischer 18090010) and the primer RKB4087 (a modified oligo d(TVN) primer) to generate cDNA reads which begin synthesis just before the poly(A) tail. This mixture was then purified using a 2x SPRI bead clean up and then followed by 2nd strand synthesis and then a 1x SPRI bead clean up. This cDNA library was then subject to PCR (NEBNExt High Fidelity 2x Master Mix) and primers RKB4089 (universal primer) and RKB4090-RKB4101 (indexed primers). The final libraries generate a broad range of fragment sizes, as such we gel extracted libraries from a 2% agarose gel corresponding to 300-500 bp size. Following extraction and purification, library size was analyzed with a 4200 TapeStation System before sequencing. RNA-seq:Poly(A)-selected, unstranded Illumina libraries were prepared following the TruSeq protocol per the manufacturer’s instructions. Library size and distribution was analyzed with a 4200 TapeStation System before sequencing.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina MiSeq
 
Description MelanA.poly(A).E
read_counts_fwd.csv, read_counts_rev.csv, pas_targets_diffapa.csv
Data processing RNA-seq was analyzed as previously described (Dvinge et al., 2014). RNA-seq reads were mapped to an annotated transcriptome created using Ensembl 71 (Flicek et al., 2013), UCSC knownGene (Meyer et al., 2013) and Misov2.0 (Katz et al., 2010) annotations using RSEM version 1.2.4(Li & Dewey, 2011) (modified to call Bowtie (Langmead et al., 2009) with option ‘-v 2’).
RNA-seq reads were mapped to an annotated transcriptome created using Ensembl 71 (Flicek et al., 2013), UCSC knownGene (Meyer et al., 2013) and Misov2.0 (Katz et al., 2010) annotations using RSEM version 1.2.4(Li & Dewey, 2011) (modified to call Bowtie (Langmead et al., 2009) with option ‘-v 2’).
Unaligned reads were then mapped to the corresponding genome (hg19/GRCh37 assembly, mm10/GRCmc38 assembly) and a database containing all possible pairings of 5’ and 3′ splice sites per gene in our merged transcriptome annotation using TopHat version 20.8b (Trapnell et al., 2009).
For Poly(A)-seq, data was mapped similarly but in a stranded fashion. BAM files were input into the APAlyzer package in R (R. Wang & Tian, 2020) and the gene level log2(distal reads / proximal reads) was computed per sample for each respective strand.
Assembly: mm10
Supplementary files format and content: csv file for Poly(A)-seq data, output of APAlyzer (Wang & Tian, 2019) as raw data per sample quantifying reads in the proximal and distal UTR isoforms per gene. tsv style table for the RNA-seq which includes the reads per gene per condition, the log-fold changes per gene and the p value.
 
Submission date Aug 29, 2022
Last update date Jun 01, 2023
Contact name Austin Max Gabel
E-mail(s) agabel1@uw.edu
Phone 4102922554
Organization name Fred Hutchinson Cancer Center
Department Computational Biology
Lab Robert K. Bradley
Street address 1100 Fairview Ave. N., B3-111
City Seattle
State/province WA
ZIP/Postal code 98109
Country USA
 
Platform ID GPL16417
Series (1)
GSE212278 Cancer-specific alternative polyadenylation shapes tumor phenotypes in vivo
Relations
BioSample SAMN30567704
SRA SRX17314126

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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