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Sample GSM1695041

Query DataSets for GSM1695041

Status

Public on Mar 09, 2016

Title

CMV_miR_122_23T_72h_rep2

Sample type

SRA

Source name

293 cells

Organism

Homo sapiens

Characteristics

cell line: 293

Extracted molecule

total RNA

Extraction protocol

Large RNA samples were homogenized and extracted by Trizol; mRNAs were enriched through poly-A purification from a poly-A spin kit (NEB); small RNA samples were extracted using a miRvana microRNA isolation kit (samples 13-50) or total Trizol-extracted RNA (samples 51-93)
RNAseq library samples 1-6 were prepared using an Illumina TruSeq kit. RNAseq library samples 7-12 were prepared using a ScriptSeq v2 kit (Epicentre).
3ug of miRvana extracted or 5ug of Trizol extracted small RNAs were ligated to Linker-1 (IDT) in the absence of ATP with T4 RNA ligase 1 (New England Biolabs). Ligated RNA was size selected (based on an 18-36nt small RNA fraction) on 15% PAGE. The 5’ ends RNA were ligated to barcoded adapters. Dual-ligated RNA was reverse transcribed, PCR amplified and size selected on 4% NuSieve (Lonza) agarose. Small RNA sequencing was performed on the Illumina GAII (samples 13-50) and miSeq (samples 51-93) platforms.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina MiSeq

Description

CMV_miR_122_matches.txt

Data processing

RNAseq reads were aligned to the mm9 genome through a standardized pipeline generated by the Stanford Center for Genomics and Personalized Medicine
FPKM values were calculated by use of Cufflinks v2.2.1 with the following parameters: “--library-norm-method quartile” and removing ribosomal, snoRNA and mitochondrial sequences.
small RNA sequencing reads were sorted by barcode, barcode removed (4nt), linker-1 clipped and aligned to mouse hairpins from mirBase release 15 using bowtie (version 0.12.7) with 2 mismatches and command -- best
small RNA sequences from 293 cells were aligned to the miR-122 plasmid that was transfected and raw counts for each small RNA that was generated were tabulated
Genome_build: mm9, mirBase release 15
Supplementary_files_format_and_content: File of FPKM values for RNAseq samples, of reads per million (RPM) mapped microRNA hits for mouse small RNA sequencing samples, and alignment to a transfected plasmid in human cell line samples, all in tab delimited text format

Submission date

May 22, 2015

Last update date

May 15, 2019

Contact name

Paul Valdmanis

E-mail(s)

paulnv@uw.edu

Phone

206-221-8059

Organization name

University of Washington