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| Status |
Public on Feb 25, 2019 |
| Title |
3'mRNAseq_zPCF11deltaPAS1_het_e2 |
| Sample type |
SRA |
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| Source name |
single embryo head 32hpf
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| Organism |
Danio rerio |
| Characteristics |
molecule subtype: 3' end of mRNA
genotype/treatment: zPCF11deltaPAS1 +/-
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| Growth protocol |
Zebrafish (Danio rerio) were raised according to standard protocols (28°C water temperature, 14/10 hr light/dark cycle). TLAB fish, generated by crossing zebrafish AB and the natural variant TL (Tupfel Long-fin) stocks, served as wild-type zebrafish for all experiments. zPCF11 null and zPCF11 ∆PAS1 mutant zebrafish were generated by Cas9-mediated mutagenesis (Gagnon et al., 2014)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Dechorionated embryos of heterozygous Pcf11-mutant incrosses were cut in half with a razor blade at 19 hpf (zPCF11null) or 32 hpf (zPCF11deltaPAS1) and, and each head and tail was collected individually in PCR tubes. The anterior halves (heads) were lysed in 10 µl of TCL buffer with 1% beta-mercaptoethanol and flash-frozen on dry ice for subsequent use for RNA isolation and sequencing. The posterior halves (tails) were used for genotyping of each individual sample. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads (A63987, Beckman Coulter).
Libraries were prepared according to the protocols of QuantSeq 3’mRNA-Seq library prep kit FW for Illumina (LEXOGEN Cat# SKU 015.96)
3-4 wild-type, 4-5 heterozygous, and six homozygous samples of both zPCF11 null and zPCF11 delta-PAS1 mutants were used for library preparation. RNA of selected samples was isolated and purified using Agencourt RNAClean XP magnetic beads (A63987, Beckman Coulter). Strand-specific libraries were generated using the QuantSeq 3' mRNA Library Prep Kit FW (Lexogen) and used for 100-bp single-end sequencing on the Illumina HiSeq 2500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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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 GRCz10/danRer10 was used as the reference zebrafish genome together with the corresponding ENSEMBL annotation).
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 annotated 3’ gene ends. 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: GRCz10/danRer10
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 |
GPL18413 |
| Series (2) |
| GSE123105 |
Selective roles of vertebrate PCF11 in premature and full-length transcript termination |
| GSE124555 |
Selective roles of vertebrate PCF11 in premature and full-length transcript termination (zebrafish 3' mRNA-seq) |
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| Relations |
| BioSample |
SAMN10679298 |
| SRA |
SRX5193223 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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