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Sample GSM1347658

Query DataSets for GSM1347658

Status

Public on Oct 17, 2014

Title

nf0h3b

Sample type

SRA

Source name

UMHF1 hTERT-immortalized fibroblasts

Organism

Homo sapiens

Characteristics

Sex: male
cell type: primary neonatal foreskin fibroblast
cell line: UMHF1
merged_sample: nf0h3a_3b_4
treatment: none
replicate: 2
sequenced molecule: nascent total RNA

Treatment protocol

Bromouridine (Bru, Aldrich) was added to the media at a final concentration of 2 mM and cells were incubated at 37°C for 30 min. Cells were then washed 3 times in PBS and harvested. When indicated, cells were pre-treated for 24 hours with 0.4 uM aphidicolin prior to Bru labeling.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated by using TRIzol reagent (Invitrogen), and Bru-labeled RNA was isolated from the total RNA by incubation with anti-BrdU antibodies (BD Biosciences) conjugated to magnetic beads (Dynabeads, Goat anti-Mouse IgG; Invitrogen) under gentle agitation at room temperature for 1 h.
Bru-labeled RNA was mixed with first strand buffer and random primers and fragmented by heating at 85 ̊C for 10 minutes. The first strand cDNA was then synthesized in the presence of Actinomycin D to result in strand specific reads. After purifying the first strand cDNA using AMPure RNAclean beads (Beckman Coulter), the second strand cDNA was synthesized. The resulting cDNA was purified with AMPure XP beads, after which the Illumina TruSeq RNA Sample Prep Kit was used to repair the cDNA ends, adenylate and ligate adaptors to the cDNA. The samples were then run on a 3% agarose gel and size-selected by excising gel slices in the 300bp region. These gel slices were purified using the QIAEX II Gel Extraction Kit (Qiagen) and then the Illumina TruSeq Kit PCR reagents were used to enrich the DNA fragments. After a final purification using AMPure XP beads, the quality and concentration of the DNA libraries were determined using an Agilent Bioanalyzer.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

segments.bed file linked to GSM1347657 (merged analysis of nf0h3a, nf0h3b, nf0h4)

Data processing

genome_build: hg19
supplementary_files_format_and_content: segments.bed = extended BED format file, one record for each HMM transcription segment, where column 4 is a unique identifier for the corresponding fused and revised transcription unit in format chrom:start-end(strand), column 5 is always 0, column 7 is the segment’s HMM state (0..9), column 8 is segment RPKM, and columns 9 is the transcription unit RPKM.
Library strategy: Bru-Seq (variant of RNA-Seq)
Call bases with Illumina Casava v1.8.2.
Subtract reads that align to the reference ribosomal DNA complete repeating unit using bowtie v.0.12.8 and parameters -n 3 -k 1 -m 1.
Align remaining reads to the reference genome using TopHat v1.4.1 and parameters –min-isoform-fraction 0, --max-multihits 1, --no-closure-search, --no-coverage-search, --bowtie-n, --initial-read-mismatches 3.
Determine fractional read coverage for every genome base using Bedtools v.2.16.2 such that a base covered by one read is recorded as having a coverage of 1/read_length.
Calculate fractional RPKM values for annotated genes, where a gene is the span of its various transcript isoforms.
Separately calculate RPKM values for 1 Kb genome bins.
Merge all samples from each cell line by summing their read counts per 1 Kb bin and recalculating bin RPKM values.
Subject merged bin RPKM values to a strand-specific Hidden Markov model (HMM) segmentation algorithm with ten logarithmically distributed output transcription states (indices 0 to 9, bed column 7).
Calculate aggregate RPKM values for each called transcription segment (bed column 8).
Empirically assign HMM transcription states of 0 to 2 a Boolean transcription state of “not transcribed” and HMM states 3 to 9 a Boolean transcription state of “transcribed”.
Fuse adjacent transcribed segments into preliminary transcription units.
Revise transcription units by comparing them to the Ensembl gene annotation such that transcription units covering at least 80% of any transcript isoform of each of two adjacent genes on the same strand were split at the most proximal transcription start site of all matching transcripts of the downstream gene.
Calculate aggregate RPKM for each fused and revised transcription unit (bed column 9).

Submission date

Mar 13, 2014

Last update date

May 15, 2019

Contact name

Thomas E. Wilson

Organization name

University of Michigan

Department

Department of Pathology

Street address

2065 BSRB, 109 Zina Pitcher