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| Status |
Public on Feb 20, 2021 |
| Title |
Asterix-FLAG eCLIP - non-crosslinked_ip |
| Sample type |
SRA |
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| Source name |
OSS cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: OSS
pull-down: Anti-FLAG M2 magnetic beads
crosslinking: none
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| Growth protocol |
Drosophila OSS (ovarian somatic sheath) cells were maintained in OSS medium (Shields and Sang M3 Insect Medium [Sigma] supplemented with approximately 5 mM potassium glutamate, 5 mM potassium bicarbonate, 10% heat-inactivated fetal calf serum [Invitrogen], 10% fly extract [Drosophila Genomics Resource Center], 2 mM reduced glutathione [Sigma], 1x GlutaMAX [Gibco], 0.01 mg/mL human insulin [Sigma], and an antibiotic-antimycotic [Gibco] consisting of penicillin, streptomycin, and Amphotericin B). Cells were cultured at ~23°C. Cultures were monitored for Mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). Mycoplasma contamination was not detected.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were grown to 80% confluency in 150 mm culture dishes. Before crosslinking, the cells were rinsed with ice-cold phosphate-buffered saline (PBS).
UV cross-linking was performed at 254 nm for ~45 seconds (400 mJ) in an HL-2000 Hybrilinker. Crosslinked cells were harvested then stored frozen at -80°C until ready to proceed.
Cell pellets were thawed in 1 mL of lysis buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, and protease inhibitors), resuspended by pipetting, then incubated on ice for 15 minutes. The cell suspension was further lysed using a Bioruptor ('low' setting at 4°C for 5 minutes [30 seconds on, 30 seconds off]).
Two microliters of Turbo DNase (2 U/μL) (LifeTech) were added to each sample as well as 10 μL of RNase I (4 U/μL) (LifeTech) and mixed by pipetting. The samples were then incubated at 37°C for exactly 5 minutes with mixing (1200 rpm in a ThermoMixer). After the five-minute incubation, the samples were placed on ice and murine RNase inhibitor (40 U/μL) (NEB) was added immediately. Cell debris was removed by centrifugation at 15,000g for 15 minutes at 4°C and the clarified supernatants were transferred to fresh tubes.
Anti-FLAG M2 magnetic beads were equilibrated in 50 mM Tris, pH 8.0, 100 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate with protease inhibitors. Fifty microliters of bead resuspension were used per sample. Beads were added to each sample and incubated with gentle agitation at 4°C for 2.5 hours. Input samples were reserved for both sequencing and Western blotting; these samples were stored at 4°C until needed. Immuno-precipitated material was isolated by magnetic separation, followed by several wash steps.
Libraries were generated essentially as described in Van Nostrand et al. (2016). Briefly, RNA ends were prepared by first treating with FastAP to remove terminal phosphates, then 5' phosphorylating using PNK. Barcoded adapters were then appended using 3' linker ligation with T4 ligase. Samples were then subjected to SDS-PAGE followed by blotting. Regions of the blot corresponding to the crosslinked protein of interest were cut out, and the RNA released from the membrane by Proteinase K treatment followed by nucleic acid extraction. Samples were reverse transcribed, RNA removed, and an additional barcoded adapter appended by 3' linker ligation. Samples were cleaned up using MyOne silane beads, then taken for final PCR amplification of the library.
Libraries were pooled in equimolar ratios according to their quantification by Bioanalyzer. Paired-end reads with two, 8-basepair, barcodes were generated on an Illumina NextSeq resulting in approximately 100 million paired-end reads (~15-20 million reads per library).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: eCLIP
Basecalling was performed with bcl2fastq2 v2.19 (Illumina).
Forward and reverse reads were joined using FLASH version 1.2.11; flash -m 25 -M 175 -O
Adapter dimers, adapter trimming, and PCR duplicate removal was performed using custom scripts, after which the reverse complement was taken to generate the original sense of the input material
Reads were mapped using STAR version 2.5.2b allowing for 50 multi-mapping sites; STAR_2.5.2b --genomeDir mm10 --runThreadN 12 --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts TranscriptomeSAM --outSAMattributes NH HI nM AS NM MD --outFilterMultimapNmax 50 --outFilterMismatchNoverReadLmax 0.05 --outFilterMismatchNmax 999
Alignment files were converted to .bed format using bedtools v2.29.0; bamtobed
Reads were annotated using custom gene annotations, filtering by class based on order of presumed biological abundance (custom scripts). rRNA > intron_28SrRNA-Psi > pseudogene_5SrRNA-Psi > pseudogene_5.8SrRNA-Psi > pseudogene_28SrRNA-Psi > pseudogene_2SrRNA-Psi > pseudogene_18SrRNA-Psi > snoRNA > snRNA > structRNA_7SLRNA > structRNA_RNaseP > structRNA_RNaseMRP > structRNA_sbRNA > structRNA_scaRNA > intron_lncRNA > lncRNA > intron_tRNA > tRNA > pseudogene_tRNA > mttRNA > intron_hpRNA > miRNA > hairpin > pri-miRNA > exon > TE_DNA > TE_LINE > TE_Satellite > TE_LTR > TE_RNA > TE_Other > TE_Unknown > TE_RC > TE_ARTEFACT > intron_SteXh > intron_Ste12DOR-RB > intron_Ste > intron_sisRNA > intron > mRNA > piRNA_cluster > intron_snmRNA > snmRNA > intron_His-Psi > pseudogene_His-Psi > pseudogene > intron_asRNA > asRNA > no_annotation
Reads counts were then adjusted to include 1/n-normalization for multimapping reads within a class (custom scripts).
Read counts were then normalized to reads per million, and pileups were calculated for each annotation (custom scripts).
Final analysis compared the various datasets to subtract background and calculate fold-enrichment scores for each annotation after thresholding for low read counts custom scripts).
Genome_build: dm6
Supplementary_files_format_and_content: *annotation_pileup_normalized.txt; Tab-delimited text files; Column 1 = annotation_class:annotation; Column 2 = 1/n-normalized read count pileup; Column 3 = 1/n-normalized read count pileup in rpm
Supplementary_files_format_and_content: Supplementary file: *_enrichment.txt; Tab-delimited text file; Column 1 = annotation; Column 2 = annotation class; Column 3 = average fold-enrichment calculated after background subtraction and normalization
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| Submission date |
May 24, 2020 |
| Last update date |
Feb 22, 2021 |
| Contact name |
Jonathan James Ipsaro |
| E-mail(s) |
jipsaro@cshl.edu
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| Organization name |
Cold Spring Harbor Laboratory
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| Lab |
Leemor Joshua-Tor
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| Street address |
1 Bungtown Road
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| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (2) |
| GSE151109 |
eCLIP of Drosophila Asterix/Gtsf1 (endogenous promoter) in Drosophila ovarian somatic sheath (OSS) cells |
| GSE151112 |
Asterix/Gtsf1 links tRNAs and piRNA silencing of retrotransposons |
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| Relations |
| BioSample |
SAMN15006174 |
| SRA |
SRX8387821 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4566723_072219_4_NXIP_annotation_pileup_normalized.txt.gz |
356.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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