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| Status |
Public on Mar 19, 2021 |
| Title |
POINTnano_HeLa_Untr_Rep2 |
| Sample type |
SRA |
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| Source name |
HeLa
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
treatment: Untreated
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| Extracted molecule |
total RNA |
| Extraction protocol |
POINT method was performed as previously described mNET-seq protocol (Nojima et al., 2016) with some alterations. In brief, crude nuclear fraction was prepared from HeLa or HCT116 cells (1x107 for POINT-seq and POINT-5, 4x107 for POINT-nano) as in mNET protocol. Chromatin pellet was resuspended in NUN1 (20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA and 50% Glycerol) and treated with modified NUN2 buffer (20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% NP-40, 1 M Urea, 3% Empigen, 1x proteasome inhibitor Complete (-EDTA) and 1x PhosSTOP). Note Empigen-treated nuclear solution was gently mixed by inverting tube a few times to avoid chromatin aggregation and incubated on ice for 10 min. The chromatin pellet was centrifuged at 400g for 30 sec. The pellet was washed with PBS twice and then digested in DNase solution (10 mM Tris-HCl (pH 7.5), 400 mM NaCl, 100 mM MnCl2, 2 U/mL RiboLock and 0.2 U/mL Turbo DNase) for 15 min. After DNA digestion, soluble digested chromatin was collected by 13,000 rpm centrifugation for 10 min. The supernatant was ten times-diluted in ice-cold NET-2E buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05 % NP-40 and 3% Empigen BB) and Pol II antibody-conjugated beads were added. 10 or 40 mg of Pol II antibody (200 or 800 mL of Dynabeads anti-mouse IgG) was used for POINT-seq and POINT-5 or POINT-nano methods. Immunoprecipitation was performed at 4oC for 1 hr. The beads were washed with 1 ml of ice-cold NET-2E buffer six times. The isolated nascent RNA was purified using Trizol reagent technology twice with once DNase treatment. Note that polyA tails were added to the isolated RNA by in vitro polyadenylation with E.coli PAP for POINT-nano. Then the pA+RNA was size-selected using SPRISelect reagent (x0.6 volume).
Library prep for illumina sequencing (NovaSeq6000) employed NEBNext Ultra II Directional RNA library prep kit and SMARTer Stranded RNA-seq kit for POINT-seq and POINT-5 analyses, respectively. For ONT sequencing, Direct cDNA library prep kit was used for POINT-nano analysis. Illumina and ONT sequencing (PromethION) were conducted by Novogene UK and the high throughput genomics team of the Wellcome Trust Centre for Human Genetics (WTCHG), Oxford.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
PromethION |
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| Data processing |
Library strategy: POINT-5
POINT-seq and POINT-5 read adaptors were trimmed using TrimGalore v0.4.4 in paired-end mode, removing reads with less than 10 bases and/or low-quality ends (20 Phred score cut-off). The resultant reads were aligned against the reference human genome (GRCh38) using STAR v2.7.0 software (Dobin et al., 2013), requiring uniquely mapped reads (--outFilterMultimapNmax 1) and minimum alignment score (--outFilterScoreMin) of 10. Additionally, for POINT-5 the 5’end of the original RNA and their directionality was extracted. To do this, the script created for mNET-seq (Nojima et al., 2015) to obtain single nucleotide resolution profiles was adapted to define the 5’end of the first read in each pair as well as its directionality.
BAM files were split by strand with SAMtools v1.9 according their bitwise flags. In POINT-seq data, forward oriented strand had 83 and 163 flags associated while reverse oriented strand had 99 and 147 flags associated. Oppositely, 99 and 147 flags for POINT-5 correspond to the forward strand, while 83 and 163 to the reverse strand. For POINT-nano, 0 and 16 flags were used to call forward and reverse strand reads, respectively. Data was visualized applying genomeCoverageBed function of BedTools to each strand independently. Trackhubs in the UCSC browser were created by employing the UCSC bedGraphToBigWig v4 tool (Kent et al., 2002).
Genome_build: hg38
Supplementary_files_format_and_content: bigWig format - read counts per bp for each strand
Supplementary_files_format_and_content: bigBed format - long reads split by strand
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| Submission date |
Oct 09, 2020 |
| Last update date |
Mar 19, 2021 |
| Contact name |
Rui Sousa-Luís |
| E-mail(s) |
rluis@medicina.ulisboa.pt
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| Organization name |
Instituto de Medicina Molecular João Lobo Antunes
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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 |
GPL26167 |
| Series (1) |
| GSE159326 |
POINT technology illuminates the processing of polymerase-associated intact nascent transcripts |
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| Relations |
| BioSample |
SAMN16407116 |
| SRA |
SRX9271733 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4826678_Forward_POINTnano_HeLa_Untr_Rep2.bb |
9.6 Mb |
(ftp)(http) |
BB |
| GSM4826678_Reverse_POINTnano_HeLa_Untr_Rep2.bb |
7.8 Mb |
(ftp)(http) |
BB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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