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| Status |
Public on Apr 23, 2015 |
| Title |
NucleoplasmRNA_DMSO |
| Sample type |
SRA |
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| Source name |
HeLa cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
mnet-seq antibody: none
treatment: DMSO
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| Treatment protocol |
HeLa cell was treated with DMSO for 4 hours.
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| Growth protocol |
HeLa cells were maintained in DMEM with 10% fetal bovine serum
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| Extracted molecule |
total RNA |
| Extraction protocol |
Nucleoplasm RNA was prepared from ~80% confluent HeLa cells in 100mm Dishes. Approximately 7x106 cells were washed with ice-cold PBS twice. The cells were lysed with ice-cold 4 ml of HLB/NP40 buffer (10 mM Tris-Hcl pH 7.5, 10 mM NaCl, 0.5% NP40 and 2.5 mM MgCl2) and incubated on ice for 5 min. After the incubation, 1 ml of ice-cold HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2 and 10 % Sucrose) was under-laid and then the nuclei were collected under 1,400 rpm centrifuge at 40C for 5 min. Isolated nuclei were resuspended in 1.25 ml of NUN1 solution (20 mM Tri-HCl pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete (Roche)) and added 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40, proteinase inhibitor 1xComplete and phosphatase inhibitor 1xPhosStop (Roche)). 15 min incubation was carried out on ice with mixing by max speed vortex for 5 sec every ~4 min and then chromatin pellets were removed under 13,000 rpm centrifuge at 4oC for 10min. Nucleoplasm supernatant was treated with 0.25 U/ml TURBO DNase (Life technologies) at 37oC for 10 min. RNA was extracted by Trizol reagent (Life technologies). This extraction steps were repeated three times.
In prior to RNA library preparations, rRNAs were depleted using Ribo-Zero rRNA removal kits (Epicentre) from 5 mg of nucleoplasm RNA. RNA was also fragmented 150-200 nt by heat treatment (94oC) for 15 min in 1xNEB first strand synthesis buffer. 200 ng of nucleoplasm RNA was used for RNA library preparations. These were carried out according to NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB). Deep sequencing using Hiseq2500 were performed by the Wellcome Trust Centre for Human Genetics (WTCHG) Oxford UK.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
nucleoplasm fraction RNA
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| Data processing |
For mNET-seq data, adaptor trimming was preformed using Cutadapt (v1.1), only keeping the reads with more than 10 bases.
mNET-seq data was then aligned to the human genome (build hg19) using TopHat (v2.0.9), with a minimum anchor length of 5 bases and allowing only for unique alignments.
For ChrRNA-seq data, alignment was preformed using TopHat (v2.0.9) with a minimum anchor length of 5 bases, allowing only for unique alignments and allowing read pairs to be separated by up to 3kb.
To aquire the last nucleotide incorporated by the polymerase in the aligned mNET-seq reads, a combination of SAMtools and inhouse python scripts was used
Then, both ChrRNA-seq and mNET-seq singe-nucleotide aligned reads were separated by strand using SAMtools, selecting the strands according to the read flag.
To make bedgraphs of the reads aligning in different strands, genomeCoverageBed (v2.17.0) was used.
After that, the values in the bedgraphs were normalized to reads per 108 sequences using an inhouse python script.
Finally, to convert the bedgraphs to bigWig, the program bedGraphToBigWig from UCSC was used.
Genome_build: hg19
Supplementary_files_format_and_content: processed data files are in bigWig format; the scores in the files represent the number of reads per 108 sequences
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| Submission date |
Dec 29, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Tomás Gomes |
| E-mail(s) |
tomasgomes@medicina.ulisboa.pt
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| Organization name |
Instituto de Medicina Molecular
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| Street address |
Av. Professor Egas Moniz
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| City |
Lisboa |
| ZIP/Postal code |
1649-028 Lisboa |
| Country |
Portugal |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE60358 |
Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing |
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| Relations |
| BioSample |
SAMN03273554 |
| SRA |
SRX825025 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1573834_NucleopRNA_DMSO_F.bw |
36.1 Mb |
(ftp)(http) |
BW |
| GSM1573834_NucleopRNA_DMSO_R.bw |
35.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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