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| Status |
Public on Apr 15, 2021 |
| Title |
1xFLAG::WAGO-3 A IP |
| Sample type |
SRA |
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| Source name |
whole worms
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| Organism |
Caenorhabditis elegans |
| Characteristics |
developmental stage: L4 staged animals
genotype: 1xFLAG::WAGO-3
experiment: IP
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| Treatment protocol |
none
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| Growth protocol |
The animals were grown on peptone rich plates with NA22 bacteria as a food source at 15C
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| Extracted molecule |
total RNA |
| Extraction protocol |
The worm extract is made from staged L4 animals grown at 15C. The protein indicated in the "title" was immunoprecipitated using anti-FLAG magnetic beads and the small RNAs bound to the protein were purified by phenol-chloroform extraction.
After the animals were collecetd, the worms were transferred to 2 ml tubes (Sarstedt, Ref: 72.693.005) with Zirconia/Silica beads (Biospec, Cat. No. 110791052) in in lysis buffer (50mM Tris-HCl ,pH 7.5, 150 mM NaCl, 1% TRITON X-100, 1 mM EDTA) supplemented with 1mM PMSF and 1 tablet of cOmplete Protease inhibitor (Roche, cat. No. 11 873 580 001) per 50 ml. The worms were lysed using MP Biomedicals Fast Prep-24 5G bead beater for 16 cycles at 8m/Sec. The lysate was centrifuged at 16100g for 10 minutes at 4°C and the supernatant was transferred to a fresh tube and the concentration was measured using standard bradford assay.
Briefly, for the Input samples, Small RNA fraction was enriched using a microRNA purification kit (Norgen biotek) with 500ng total RNA as input and for IP samples, this step was excluded. The small RNA fraction was treated with 20U of RNA 5’ Polyphosphatase (TAP, Lucigen), in a 50µl reaction at 37°C for 1h. TAP-treated RNA was purified using the Norgen Biotek Single Cell RNA Purification kit and eluted in 10µl. 5µl was used as input for small RNA library preparation using the QIAseq miRNA library kit (Qiagen) according to the manufacturer recommendations. Libraries were sequenced on the Illumina HiSeq 2500 (50cycles single-end run).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
With the aid of custom scripts that rely on HTSeq (version 0.11.2), pandas (version 1.0.1) and BioPython (version 1.7) we keep the reads that contain at least 12 bases after the 3’adapter (AACTGTAGGCACCATCAAT)
The 3’ adapters were removed with cutadapt (version 2.3) (--error-rate 0.1, --minimum-length 15, --overlap 3)
Low quality reads were filtered with fastq_quality_filter from FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) (version 0.0.14) (-q 20, -p 100, -Q 33)
Reads were mapped to the genome with bowtie (version 1.2.3) (-v 0, --all, --best, --strata) and converted to bam files with samtools (version 1.9).
A custom script was used to filter reads that fall in transcripts annotated (transcript_biotype) as rRNA, rRNA_pseudogene, tRNA, tRNA_pseudogene. Reads from MtDNA were also filtered.
Filtered reads were converted back to fastq files with a custom script and reads were mapped again to the genome with bowtie (-v 0, --all, -M 1, --best, --strata)
htseq-count from HTSeq was used to count reads that fall in the opposite strand of reads/repeats (--stranded reverse, --type exon, --idattr gene_id, --mode union, -a 0, --nonunique none, --secondary-alignments ignore, --supplementary-alignments ignore)
Genome_build: ce11
Supplementary_files_format_and_content: tsv file with counts
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| Submission date |
Jun 03, 2020 |
| Last update date |
Apr 15, 2021 |
| Contact name |
Foivos Gypas |
| E-mail(s) |
[email protected]
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| Organization name |
FMI
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| Lab |
Grosshans
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| Street address |
Maulbeerstrasse 66
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| City |
Basel |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL18245 |
| Series (2) |
| GSE151716 |
Protease-mediated processing of Argonaute proteins controls small RNA association [RIP-Seq] |
| GSE151717 |
Protease-mediated processing of Argonaute proteins controls small RNA association |
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| Relations |
| BioSample |
SAMN15095236 |
| SRA |
SRX8464093 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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