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Sample GSM1518091

Query DataSets for GSM1518091

Status

Public on Apr 01, 2015

Title

siPP1α.3

Sample type

SRA

Source name

knock down PP1α in proliferating C2C12 cells

Organism

Mus musculus

Characteristics

cell line: C2C12
treatment time: 48 hr
kd batch id: 3

Treatment protocol

Differentiated C2C12 cell samples in this study are those harvested four days after differentiation. Transfection with siRNAs was carried out by Lipofectamine™ 2000 (Invitrogen) according to manufacturer’s recommendations. Transfection was carried out for 48 hr or 32 hr. The U1D oligo (5'-gCcAgGuAaGuau) and mutant U1D (mU1D) oligo (5'-gCcAgGcAcGuau), where locked nucleic acid (LNA) residues are in uppercase and 2’-OMe RNA bases are in lowercase, were previously described in Goraczniak et al. (Goraczniak et al., 2009). These oligos were transfected into C2C12 cells at 35 μM using Lipofectamine 2000 when the confluency of cells was about 50%. Cells were harvested 8 hr or 24 hr after transfection.

Growth protocol

C2C12 cells were maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Differentiation of C2C12 cells was induced by switching cell media to DMEM+ 2% horse serum (Sigma) when cells were ~100% confluent. All media were also supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin.

Extracted molecule

total RNA

Extraction protocol

For nuclear RNA extraction, cells collected by a scrapper were suspended in cell lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 0.5% NP40, 1 mM DTT), followed by vortexing for 10 sec and incubation on ice for 10 min. After centrifugation of the lysate at 500 x g for 5 min at 4oC, the pellet was re-suspended in the cell lysis buffer for nuclear RNA extraction. Both total and nuclear RNAs were extracted using Trizol (Invitrogen) according to manufacturer's protocol. RNA quality was analyzed in an Agilent Bioanalyzer using the RNA pico600 kit before processing for deep sequencing.
The 3’READS method used in this study was previously described (Hoque et al., 2014). Briefly, 25 μg of total RNA was used for each sample, and poly(A)+ RNA was selected using oligo d(T)25 magnetic beads (NEB), followed by on-bead fragmentation using RNase III (NEB). Poly(A)+ RNA fragments were then selected using the chimeric U5 and T45 (CU5T45) oligo conjugated on streptavidin beads, followed by RNase H digestion (NEB). Eluted RNA fragments were ligated with 5’ and 3’ adapters, followed by RT and PCR (15x) to obtain cDNA libraries for sequencing on the Illumina platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

The details of the method was described previously (Hoque, et al., 2014). Briefly, the 3'-adapter sequence was first removed (using fastx_clipper -a TGGAATT). Reads were mapped to the mouse genome (mm9) using Bowtie2 (local mode). 5' four random nucleotides were removed before mapping using "-5 4" parameter in bowtie2. Reads with a mapping quality score (MAPQ) ≥10 were selected for further analysis. Reads with ≥2 non-genomic As after alignment were called polyA site supporting (PASS) reads.
All PASS reads mapped to the 3’-most exons of a gene were used to calculate the expression level of the gene, shown as the reads per million total PASS reads (RPM) value.
Genome_build: mm9
Supplementary_files_format_and_content: tab delimited file containing information of gene symbol, pA coordinates, read number of pAs in different samples

Submission date

Oct 02, 2014

Last update date

May 15, 2019

Contact name

Wencheng Li

Organization name

PTC Therapeutics

Street address

100 Corporate Count

City

South Plainfield