GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3318276

Query DataSets for GSM3318276

Status

Public on Jun 10, 2019

Title

Nascent_RNA-seq_brain_biologicalRep1

Sample type

SRA

Source name

Brain

Organism

Mus musculus

Characteristics

tissue: brain
urea treatment: 2M
strain: C57BL/6NJcl
age: 8 weeks
molecule subtype: Nascent RNA

Treatment protocol

For the time course experiment, MCF-7 cells were serum-starved for 16 hours, and heregulin beta-1 was added to the medium at a final concentration of 10 nM. Cells were harvested at 0 (no treatment), 15, 30, 45, 60, 120, 180 and 360 minutes in triplicates.

Growth protocol

MCF-7, HeLa-S3 and HepG2 cells were cultured at 37 ˚C under 5% CO2 in DMEM with L-Gln supplemented with 1 mM sodium pyruvate and 10% FBS. GM12878 and K562 cells were cultured at 37 ˚C under 5% CO2 in RPMI 1640 with L-Gln supplemented with 1 mM sodium pyruvate and 10% FBS.

Extracted molecule

total RNA

Extraction protocol

The cDNA synthesis was carried out using 5 μg of total and nascent RNA (A260/280 and 260/230 ratios > 1.7). CAGE libraries were generated based on no-amplification non-tagging CAGE libraries for Illumina next-generation sequencers (nAnT-iCAGE) precisely as described in Murata et al. (Methods Mol Biol. 2014;1164:67-85.).
Frozen mouse tissue samples were used for total and nascent RNA extraction. Tissue was homogenized using gentleMACS Dissociator (Miltenyi Biotec). The sample was transferred to gentleMACS M tube (Miltenyi Biotec) and added with pre-chilled 2 ml of Buffer C (Nuclei PURE Lysis Buffer (Sigma-Aldrich), 25 μM alpha-amanitin, 1× cOmplete Protease Inhibitor solution, 20 units of SUPERase IN RNase Inhibitor, 1 mM DTT, Triton X-100). Lysate was then filtered through gentleMACS SmartStrainers (100 μm) (Miltenyi Biotec). 200 μL of the tissue lysate was sampled and mixed with 700 μL of QIAzol for total RNA extraction.
We added 2 ml of ice-cold Buffer D (10 ml of Sucrose Cushion solution (Sigma-Aldrich), 1.2 ml of Sucrose Cushion buffer (Sigma-Aldrich) and 11.2 μL of 1M DTT) to the bottom of a conical centrifuge tube on ice. Tissue lysate prepared above was gently mixed with 3.6 ml of Buffer D, and was carefully and slowly layered on top of the 2 ml of Buffer D in the conical centrifuge tube. Then, the tube was centrifuged at 13,000 G for 45 minutes at 4 ˚C to collect nuclei pellets. The pellets were washed once with 500 μL of Buffer C.
Washed pellets were resuspended in 200 μL of Buffer B (1% NP-40, 20 mM HEPES, 300 mM NaCl, 2 M Urea, 0.2 mM EDTA, 1 mM DTT, 25 μM alpha-amanitin, 1× cOmplete Protease Inhibitor solution and 20 units SUPERase IN RNase Inhibitor), and incubated for 10 minutes on ice. Then, nuclear insoluble fraction was collected by 3,000 G centrifuge for 2 minutes at 4 ˚C. The pellets were washed once with 100 μL of Buffer B.
50 μL of DNase I solution containing 10 units of DNase I (Thermo Fisher Scientific), 1× DNase I Buffer (Thermo Fisher Scientific) and 20 units of SUPERase IN RNase Inhibitor was added to the pellets. The solution was incubated for 30 minutes at 37 ˚C while pipetting up and down several times every 10 minutes. 700 μL of QIAzol was directly added to the solution and thoroughly mixed. RNA was extracted by miRNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. An “on-column DNase I digestion” was carried out using RNase-free DNase set (QIAGEN). RNA was eluted in 30 μL of RNase-free water, and quality and quantity were measured by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and 2100 BioAnalyzer (Agilent).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

NET_Brain

Data processing

For all our datasets we performed the following steps, (i) split reads by barcode, (ii) trimmed reads to remove barcode sequences and (iii) removed reads aligned to rRNA sequences (Human: GenBank U13369.1; Mouse: GenBank BK000964.3). These steps were performed using MOIRAI (Hasegawa et al., 2014) for human cell line data and using Trimmomatic v 0.36 (Bolger et al., 2014) and MOIRAI (Hasegawa et al., 2014) for mouse tissue data. For all CAGE based data and mouse RNA-seq data, sequences with base ‘N’ were also removed.
Non rRNA reads were mapped hg19 genome and human reference gene model Gencode v27lift37 comprehensive using STAR v 2.5.0a with the following parameters for indexing: --runThreadN 12 --runMode genomeGenerate --genomeDir genome.fa --sjdbGTFfile Gencodeannotation.gtf and the following parameters for mapping: --runThreadN 12 --outSAMtype BAM SortedByCoordinate --out FilterMultimapNmax 1 --readFilesCommand zcat.
Transcription start sites for CAGE based data were identified using bedtools v 2.2.25 (Quinlan and Hall, 2010) as described here: http://fantom.gsc.riken.jp/5/sstar/Protocols:HeliScopeCAGE_read_alignment
Genome_build: Human: hg19 and Mouse:mm9
Supplementary_files_format_and_content: Bed 6 file for trasncription start sites. Scores represent aggregated tags with same starting position.

Submission date

Aug 03, 2018

Last update date

Jun 10, 2019

Contact name

Shruti Bhagat

E-mail(s)

shruti.bhagat@riken.jp

Organization name

RIKEN, Yokohama

Department

RIKEN Center for Integrative Medical Sciences