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| Status |
Public on Oct 04, 2014 |
| Title |
HeLa GRO-Seq rep1 |
| Sample type |
SRA |
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| Source name |
HeLa cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
gro-seq antibody: anti-BrU (sc-32323-ac)
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| Treatment protocol |
no special treatments of cells were performed for this study
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| Growth protocol |
HeLa cells were grown in DMEM media supplemented with 10% FBS and 1x Pen/strep.
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| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate nuclei, cells were re-suspended in 5 ml LYSO-A buffer (35 mM Tris-Cl, pH7.5, 150 mM sucrose, 80 mM KCl, 5 mM MgCl2, 0.5 mM DTT) and centrifuged for 5 min at 1000 g. They were then re-suspended in 5 ml LYSO-A buffer containing 0.05% Tween-20. Cells were incubated on ice for 1-5 min, with constant checking to ensure that 90% of cells were permeabilised, using a haemocytometer and tryphan blue (Life Technologies). Cells were washed in 5 ml LYSO-A buffer and transferred to siliconized tubes (Eppendorf) in 1 ml LYSO-A. Cells were centrifuged at 900 g for 5 min and re-suspended in 500 μl F80 buffer (50 mM Tris, pH8.3, 40% glycerol, 80 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT). Cells were counted and then re-suspended at 5 x 106 cells/100 μl before being snap-frozen in liquid nitrogen in aliquots of 100 μl. All steps were carried out at 4°C and using wide-bore pipette tips to minimize the loss of sample.
GRO-seq libraries were prepared as in Core et al. (Core, Waterfall, and Lis, 2008), with the following modifications. Trizol (invitrogen) was used to stop the reaction instead of DNase I and proteinase K treatment. The RNA was further extracted once with acid phenol:chloroform, and once with chloroform before precipitating with 2.5 volumes of -20oC ethanol. Bead binding buffers all contained 4units/ml of SUPERaseIN (ambion) and the following buffers were slightly modified. Bead blocking buffer: 0.25X SSPE, 1mM EDTA, 0.05% tween, 0.1% PVP, and 1mg/ml ultrapure BSA (Ambion); Binding buffer: 0.25XSSPE, 37.5mM NaCl, 1mM EDTA, 0.05% tween; Low salt wash buffer: 0.2X SSPE, 1mM EDTA, 0.05% Tween. High Salt wash buffer: 0.25% SSPE, 137.5mM NaCl, 1mM EDTA, 0.05% Tween. The end repair steps were modified as follows. Pelleted RNA from the first bead binding was resuspended in 20ul, and heated to 70oC for 5min, followed by incubation on ice for 2min. 1.5ul tobacco acid pyrophosphatase (TAP) buffer, 4.5ul water, 1 ul SUPERaseIn, and 1.5ul TAP (Epicentre) were then added and the reaction incubated at 37oC for 1.5 hours. 1ul 300mM MgCl2 and 1ul T4 polynucleotide Kinase (PNK) were added to the reaction for an additional protocols l 30 min. for phosphorylating the 5’-ends, 20ul T4 PNK buffer, 2ul 100mM ATP, 145ul water, 1ul SUPERaseIn, and an additional 2ul of PNK were added for 30 min at 37oC. The reaction was then stopped by addition of 20mM EDTA followed by acid phenol extraction and precipitation.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
nascent RNA
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| Data processing |
Library strategy: GRO-seq
Pass Filter: Reads that pass filter were selected from fastq files.
Read Trimming: reads were trimmed to 30 bases
rDNA filter: Reads were first mapped to a copy of rRNA gene (GenBank accession #: U13369.1) including external and interanl spacers, and only reads that were not mapped to the rDNA were kept.
mapping to the genome: Bowtie was used to map 30mers with up to two mismatches to the mm9 genome.
Genome_build: hg19
Supplementary_files_format_and_content: Processed files are Bedgraphs from combined replicates. Each entry represents the number of reads at each base.
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| Submission date |
Oct 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Robin Andersson |
| E-mail(s) |
robin@binf.ku.dk
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| Organization name |
University of Copenhagen
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| Street address |
Ole Maaloes Vej 5
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| City |
Copenhagen N |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE62046 |
GRO-seq in HeLa cells |
| GSE62047 |
Nuclear stability and transcriptional directionality separate functionally distinct RNA species. |
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| Relations |
| Reanalyzed by |
GSE66031 |
| Reanalyzed by |
GSE85747 |
| BioSample |
SAMN03093227 |
| SRA |
SRX719733 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1518913_HeLa_rep1.minus.bedgraph.gz |
18.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1518913_HeLa_rep1.plus.bedgraph.gz |
20.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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