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Sample GSM6523080

Query DataSets for GSM6523080

Status

Public on Jun 01, 2023

Title

B16-F10 cells poly(A) seq E

Sample type

SRA

Source name

B16-F10

Organism

Mus musculus

Characteristics

cell line: B16-F10
cell type: melanoma
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

B16-F10.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