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| Status |
Public on Jun 10, 2019 |
| Title |
CAGE_MCF-7_time360_biologicalRep1 |
| Sample type |
SRA |
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| Source name |
Breast
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| Organism |
Homo sapiens |
| Characteristics |
tissue: MCF-7 cell line
urea treatment: NA
molecule subtype: Total RNA
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| 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.
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| 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.
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| 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.).
Approximately 1.0 × 107 cells were used for total and nascent RNA fractionation. Cultured medium was aspirated and cells were gently washed twice with 5 mL of ice-cold 1x D-PBS. To lyse cells, 1,400 μL of Buffer A (Nuclei ez lysis buffer (Sigma-Aldrich), 25 μM alpha-amanitin (FUJIFILM Wako Pure Chemical), 1× cOmplete Protease Inhibitor solution (Sigma-Aldrich) and 20 units of SUPERase IN RNase Inhibitor (Thermo Fisher Scientific)) was directly added to a dish. Cells treated with Buffer A were harvested using a cell scraper. 100 μL of the cell lysate was sampled and mixed with 700 μL of QIAzol (QIAGEN) for total RNA extraction. The remaining lysate was set on ice for 10 minutes. Then the lysate was centrifuged at 800 G for 5 minutes at 4 ˚C. The pellets were washed once with 200 μL of Buffer A.
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).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
CNhi10945_NoIndex_L003_R1_001_GCT
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| Data processing |
Library strategy: RNA-seq (CAGE)
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.
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| Submission date |
Aug 03, 2018 |
| Last update date |
Jun 10, 2019 |
| Contact name |
Shruti Bhagat |
| E-mail(s) |
shruti.bhagat@riken.jp
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| Organization name |
RIKEN, Yokohama
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| Department |
RIKEN Center for Integrative Medical Sciences
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| Lab |
Preventive Medicine and Applied Genomics Unit
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| Street address |
W424, RIKEN Yokohama Campus, 1-7-22 Suehiro-cho, Tsurumi-ku
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| City |
Yokohama |
| State/province |
Kanagawa |
| ZIP/Postal code |
230-0045 |
| Country |
Japan |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE118075 |
NET-CAGE Characterizes the Dynamics and Topology of Human Transcribed Cis-regulatory Elements |
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| Relations |
| BioSample |
SAMN09763217 |
| SRA |
SRX4504712 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3318157_CNhi10945_biologicalRep1-MCF7-CAGE.Time360_GCT.ctss.bed.gz |
9.8 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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