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Sample GSM758325

Query DataSets for GSM758325

Status

Public on Feb 03, 2014

Title

h9ESC short 1

Sample type

SRA

Source name

Embryonic stem cells

Organism

Homo sapiens

Characteristics

developmental stage: Embryonic stem cells
cell line: H9

Growth protocol

The hESC H9 was grown on MEF feeders (2 Ã 104 cells/cm2) in DMEM/F12 medium plus 20% Knockout Serum Replacement (Invitrogen, Carlsbad, CA) and bFGF (4 ng/ml, Sigma-Aldrich, St. Louis, MO).

Extracted molecule

total RNA

Extraction protocol

10ug total RNA was harvested from hESC H9 by Tri-reagent (Ambion) for cDNA library preparation. The enrichment of mRNA by depleting ribosomal RNA was performed by RiboMinusTM Transcriptome isolation Kit (Invirogen) according to manufacturerâs instruction. One ug RiboMinus RNA was fragmented by treating RNase III 10 minutes and cleaned up by RiboMinus Concentration Module (Invitrogen). The fragmented RiboMinus RNA was ligased with SOLiD adaptor A and reverse transcribed by ArrayScript Reverse Transcriptase. The product of reverse transcription was purified by MinElute PCR Purification Kit (Qiagen) and size-selected on 6% TBE âUrea Gel for a size range from 100nt to 200nt. The cDNA library with proper size was amplified by SOLiD PCR Kit. To prepare the sequencing template, size selected cDNA library was coupled with SOLiD P1 DNA beads and mixed with emulsion PCR mixture by ULTRA-TURRAX Tube Drive from IKA. The emulsion PCR was performed by GeneAmp PCR system 9700 according to manufacturerâs program. The templates on SOLiD P1 DNA beads amplified by emulsion PCR were washed, denatured and enriched by SOLiD P2 bead incubation. The enriched templates were then modified at the 3â end with bead linker by terminal transferase reaction. The 3â end modified templates were washed and deposited to SOLiD slide. Sequencing of templates were performed by SOLiD 3 instrument according to the operation guide. Data was processed with SOLiD Analysis Tool Pipeline.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

AB SOLiD System 3.0

Description

RiboMinus Transcriptome isolation, Strand specific

Data processing

We aligned the h9ESC SOLiD reads and the h1ESC Illumina reads (downloaded from GSE20301) against the non-co-linear junction regions of the 9,093 candidates using BFAST. For each non-co-linear junction region, both of the non-overlapping regions of mapping parts are restricted to being longer than 10 bp in length and showing greater than or equal to 95% alignment to the mapping short reads. Only one mismatch and no insertion/deletion are allowed in a mapping between a non-co-linear junction region and a short read. A matched short read is discarded if it can also map to the human genome or well-annotated transcripts (including UCSC- and Ensemble-annotated transcripts). After the filter of short reads, TSscan nominates 47 non-co-linear RNA candidates. The mapping results of short reads against the non-co-linear junction regions are also provided (i.e., mapping.rar).

Submission date

Jul 11, 2011

Last update date

May 15, 2019

Contact name

Chan-Shuo Wu

E-mail(s)

chanshuo@gate.sinica.edu.tw

Organization name

Academia Sinica

Department

Genomics Research Center