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| Status |
Public on Feb 25, 2019 |
| Title |
3'mRNAseq_siPCF11_rep1 |
| Sample type |
SRA |
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| Source name |
HeLa cells
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| Organism |
Homo sapiens |
| Characteristics |
molecule subtype: 3' end of mRNA
genotype/treatment: PCF11 knock-down (5 nM)
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| Treatment protocol |
siRNAs from Dharmacon were transfected for 48 hrs according to RNAiMAX reagent protocol at the concentrations indicated.
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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 |
Nuclei were prepared by incubation of cells 5 mins on ice in HLB (10 mM Tris pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 0.5% NP40), underlayering with HLB+NS buffer (10 mM Tris pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 0.5% NP40, 10% sucrose) and centrifugation 420 g for 5 min at 4oC. RNA was purified from the nuclei using TRI reagent (Sigma) according to manufacturer’s instructions. Residual DNA was digested using 4U Turbo DNase (Life Tech) for 10 min at 37oC followed by proteinase K digestion for 10 min at 37oC. TRI reagent purification and DNase digestion were repeated. RNA was further acid phenol/chloroform and chloroform extracted, followed by ethanol precipitation. The purified RNA was then resuspended in ultrapure water.
Libraries were prepared according to the protocols of QuantSeq 3’mRNA-Seq library prep kit REV for Illumina (LEXOGEN Cat# SKU 016.24)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
nuclear RNA
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| Data processing |
3’ mRNA-seq data were mapped according to the guidelines of QuantSeq library kit manufacturer (Lexogen). Unaligned bam files from HiSeq2500 were converted to FASTQ files with bam2fastx (TopHat 2 component (Kim et al., 2013)). After overview with FastQC reads were trimmed with BBtools (sourceforge.net/projects/bbmap/) script bbduk using following settings: k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20 .
After trimming control with FastQC curated reads were mapped with STAR2.5b (Dobin et al., 2013) aligner. STAR index was generated with the GRCh37.p13 assembly and gencode.v19.annotation.gtf annotation file).
Aligned 3’ mRNA-seq reads were filtered to remove false positives due to internal priming of the QuantSeq assay on genome-encoded poly(A) stretches. To do this, first a crude genomic mask was generated that contained all loci harbouring 6 or more consecutive A bases as well as any 10 nucleotide windows containing more than 6 A bases. For genes expressed from the reverse strand, an analogous T-rich mask was generated. Those crude masks were then corrected to allow for detection of genuine PAS falling in A/T-rich regions by strand-specifically unmasking 20 nucleotide intervals centred at GENCODE 3’ gene ends as well as previously experimentally validated PAS sites detected in all human data sets from (Derti et al., 2012). 3’ mRNA-seq reads falling into those refined strand-specific masks were then removed, and the filtered reads reduced to the most distal nucleotide (3’ end nucleotide).
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files: genome-coverage files, mean from replicates
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| Submission date |
Jan 02, 2019 |
| Last update date |
Feb 25, 2019 |
| Contact name |
Kinga Kamieniarz-Gdula |
| Organization name |
University of Oxford
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| Department |
Sir William Dunn School of Pathology
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3RE |
| Country |
United Kingdom |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE123105 |
Selective roles of vertebrate PCF11 in premature and full-length transcript termination |
| GSE124556 |
Selective roles of vertebrate PCF11 in premature and full-length transcript termination (human 3' mRNA-seq) |
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| Relations |
| BioSample |
SAMN10679993 |
| SRA |
SRX5193237 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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