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| Status |
Public on Apr 23, 2015 |
| Title |
mNET_8WG16_siCPSF73 |
| 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: 8WG16
treatment: CPSF73 siRNA
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| Treatment protocol |
SMARTpool siRNA against human CPSF73 (CPSF3) (final conc. 30nM) were transfected into HeLa cell using Lipofectamine RNAiMAX reagent (Life technologies) according to the manual and incubated for 72 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 |
Approximately 1.6x108 cells were used to generate nuclear and chromatin fractions. Isolated chromatin was washed in 1 ml of 1x Micrococcal Nuclease (MNase) buffer (NEB) and then incubated with MNase (40 u/mL) on Thermomixer (eppendorf, 1,400 rpm) at 37oC for 90 sec. MNase was inactivated by EGTA (25 mM) addition immediately after the reaction and soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. The supernatant was diluted with 9 ml of NET-2 buffer and add Pol II antibody-conjugated beads. 40 mg of Pol II antibody was used for each mNET-seq experiment. Immuno-precipitation was performed at 4oC for one hour. The beads were washed with 1 ml of NET-2 buffer six times and with 500 ml of 1xPNKT (1xPNK buffer and 0.05 % Triton X-100) buffer once in the cold room. The washed beads were incubated in 100 ml of PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK 3'phosphatase minus (NEB) ) on Thermomixer (1,400 rpm) at 37oC for 6 min. After the reaction the beads were washed with 1 ml of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was resolved on 8 % denaturing acrylamide 7 M urea gels for size purification. 35-100 nt fragments were eluted from the gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) and RNA was precipitated in 75 % Ethanol.
RNA libraries were prepared according to the manual of Truseq small RNA library prep kit (Illumina). Deep sequencing was conducted by WTCHG in Oxford.
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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 from unphosphorylated Pol II
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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 |
SAMN03273557 |
| SRA |
SRX825030 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1573839_mNET_8WG16_siCPSF73_F.bw |
33.1 Mb |
(ftp)(http) |
BW |
| GSM1573839_mNET_8WG16_siCPSF73_R.bw |
32.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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