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Sample GSM1269427

Query DataSets for GSM1269427

Status

Public on May 26, 2014

Title

sD3N_int_ret

Sample type

SRA

Source name

Nuclear

Organism

Homo sapiens

Characteristics

cell line: HEp-2 cells
analysis type: Intron retention
treatment: Dis3 knockdown (Nuclear)

Treatment protocol

1.8E6 cells were seeded in T175 flasks 24 h prior to transfection. 750 pmol siRNA was transfected using Oligofectamine (Invitrogen) according to manufacturer's protocol.

Growth protocol

Cells were grown in DMEM supplemented with 10% FBS and antibiotics in humidified incubator with 5% CO2 with regular passaging.

Extracted molecule

total RNA

Extraction protocol

Cells were trypsinized, washed once with cold PBS, resuspended in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0,5 mM DTT, protease inhibitors) and incubated on ice 10 min. Then cells were spun down, resuspended in hypotonic buffer, and lysed with Kontes tight pestle (B) douncer. Lysis was monitored under the microsocpe with Trypan blue. Lysate was layered over 1 M sucrose in hypotonic buffer and spun 5000 rpm 30 min 4 °C, which pelleted the nuclei at the bottom of sucrose cushion. Nuclei were washed once with sucrose hypotonic buffer. Samples were sonicated 3 x 30 sec using Bioruptor sonicator, and cleared by 10 min 13 000 rpm centrifugation in an Eppendorf centrifuge.
Sequencing library was constructed from fractionation pools using SOLiD Total RNA-Seq Kit (Applied Biosystems). Briefly, library construction contained the following steps: ribosomal RNA removal, fragmentation using RNaseIII, reverse transcription, amplification (14 cycles), and size selection (70-150 bp).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

AB SOLiD 4 System

Description

For the Intron retention analysis, the read (single or pairs) that were fully mapped to the introns or exons of the U12 transcripts were taken into account.
u12_transcripts_Intron_retention.tsv

Data processing

Mapping: Mapping of the sequence reads were done using SOLiD Bioscope V1.3
Extracting unique reads: PCR duplicates were removed.
Summarization: Reads that were mapped to introns or exons of transcripts with at least one U12 intron were counted. For the Intron retention analysis, the fragments that were fully mapped to the introns or exons of the transcripts were taken into account (see u12_transcripts_Intron_retention.tsv). Furthermore, reads mapped to the Intron-exon junctions (see u12_transcripts_Intron_exon_junction.tsv) of U12 transcripts and also intron retentions of the U2 introns from randomly picked transcripts that feature U2 type introns only (see u2Random_transcripts_Inron_retention.tsv) were estimated through separate analysis.
Normalization: FPKM values were calculated by normalizing the number of fragments to the length of the introns/exons and to the number of fragments that were mapped to the transcript in order to remove the bias of intron/exon length and transcript expression.
Differentiation analysis: Log-fold change of the FPKM values together with the edge-R Pvalues were calculated to estimate the significance of changes in intron retention across ctrl (i.e. sGN) and knock-downs (i.e. s41N and sD3N).
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file including annotatuion of Introns or exons of U12 transcripts,read counts and FPKM values for Nuclear (sGN) and Cytoplasmic ctrl (sGC) and samples in which the exosome was deactivated by knocking down Rrp41 (s41N for nuclear and s41C for cytoplasmic) and DIS3 (sD3N and sD3C) subunits.

Submission date

Nov 19, 2013

Last update date

May 15, 2019

Contact name

Mikko Juhani Frilander

Organization name

Institute of Biotechnology

Department

University of Helsinki

Lab

RNA-splicing laboratory