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Sample GSM3060930

Query DataSets for GSM3060930

Status

Public on Mar 23, 2018

Title

2011-395 RNA142

Sample type

SRA

Source name

corneal endothelium

Organism

Homo sapiens

Characteristics

group: FECD NO expansion
batch: 3
re: RE-

Extracted molecule

total RNA

Extraction protocol

RNA libraries were prepared using TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA). Briefly, poly(A) mRNA was purified from total RNA using oligo(dT) magnetic beads, fragmented at 95 °C for 8 min, eluted from the beads, and primed for first-strand cDNA synthesis using SuperScript III reverse transcriptase and random primers (Invitrogen). Second- strand cDNA synthesis was performed using DNA polymerase I and RNase H, and double-stranded cDNA was purified using a single AMPure XP bead cleanup step (Agencourt, Danvers, MA). The cDNA ends were repaired and phosphorylated using Klenow fragment, T4 polymerase, and T4 polynucleotide kinase, followed by a single AMPure XP bead cleanup step. The blunt-ended cDNAs were modified to include a single 3 -adenylate residue using Klenow exo (3 to 5 exo ), and paired-end DNA adaptors (Illumina) with a single T base overhang at the 3 -end were ligated to the A-tailed cDNA population. Unique indexes, included in the standard TruSeq kits (12- Set A and 12-Set B), were incorporated at the adaptor ligation step for multiplex sample loading on the flow cells. The resulting constructs were purified by two consecutive AMPure XP bead cleanup steps and enriched by 12 cycles of PCR using primers included in TruSeq RNA Sample Prep Kit v2. Libraries were loaded onto paired-end flow cells at concentrations of 8–10 pM to generate cluster densities of 700,000/mm2 using cBot and cBot Paired-end Cluster Kit v3 (Illumina) following the manufacturer’s standard protocol. The flow cells were sequenced as 51 2 paired-end reads on an Illumina HiSeq 4000 system using TruSeq SBS Sequencing Kit v3 and SCS v1.4.8 data collection software.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

RNA142

Data processing

Base calling Illumina CASAVA v1.8.2
QC was performed on the FASTQ files using RSeQC software (http://rseqc.sourceforge.net/)
The sequencing reads were processed by MAP-RSeq v.1.2 workflow [1], the Mayo Bioinformatics Core pipeline. The alignment in MAPRseq was performed with TopHat 2.0.6 and the gene counts was obtained using HTSeq-count v0.5.3p9
Genome_build: hg19
Supplementary_files_format_and_content: tab delimited file includes raw gene counts

Submission date

Mar 22, 2018

Last update date

Mar 23, 2018

Contact name

Xiaojia Tang

E-mail(s)

Tang.Xiaojia@mayo.edu

Organization name

Mayo Clinic

Street address

200 1st ST SW