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| Status |
Public on Feb 05, 2021 |
| Title |
W-PUT-2 |
| Sample type |
SRA |
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| Source name |
Input lysate, Heads
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| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: elav>Gal4
genotype (shorthand): W
sample/treatment type and description: PUT; RNA from Input samples
tissue: decapitated adult female heads, isolated in bulk protocol
tissue amount: 300 heads
control or experimental?: Control
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| Treatment protocol |
300 female Drosophila heads each of the genotypes elav>Gal4 alone, elav>Nab2-FLAG, and elav>Atx2-3xFLAG, previously isolated in bulk (see Supplementary Materials and Methods in the Rounds et al. submission associated with this accession), were fixed in 1% formaldehyde, 0.1% NP-40 in PBS for 30 minutes at 4°C. Fixation was quenched by adding glycine to a final concentration of 250 mM and rocking for 10 minutes at 4°C. Heads were washed in 0.1% NP-40 in PBS and then manually homogenized with a smooth Teflon pestle for 5 minutes in 250 µL of NIB in a size AA glass tissue grinder at 4°C (3431D70, Thomas Scientific). Homogenates were spun through 35 µm cell strainer caps into round-bottom tubes (352235, Falcon) to remove exoskeletal debris, transferred, and then centrifuged for 5 minutes at 500×g at 4°C to separate an insoluble fraction. Twenty percent of the soluble supernatant volume was isolated and defined as Input; the remaining eighty percent was used for immunoprecipitation. Both Input and IP samples were diluted to final concentrations of 0.8x IP Buffer to ensure comparable and efficient sample lysis. IP samples were transferred onto the α-FLAG-conjugated magnetic Dynabeads, and both sample types were incubated, rotating, for 10 minutes at room temperature. Next, IP sample supernatant was collected as the Unbound fraction, and IP sample beads were washed three times in IP Buffer. Finally, IP sample beads were resuspended in IP Buffer, transferred to clean tubes, and stored along with Input samples overnight at 4°C to allow passive hydrolysis to partially reverse formaldehyde crosslinks. For RNA immunoprecipitation, harsh elution of RNA from IP sample beads was accomplished the next day with Trizol—both IP and Input samples were subjected to the RNA extraction protocol detailed below.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Following immunoprecipitation, RNA was isolated from IP and Input samples using a TRIzol-column hybrid approach adapted from (Rodriguez-Lanetty). To account for volume differences, samples were vigorously homogenized in TRIzol reagent (15596018, Thermo Fisher) at a ratio of either 1:10 (IP sample:TRIzol) or 1:3 (Input sample:TRIzol) and then incubated for 5 minutes at room temperature. All homogenized samples were clarified by centrifugation at 12,000×g at 4°C for 5 minutes, IP samples were magnetized to collect beads, and supernatant was isolated from all samples. After adding chloroform at a ratio of 0.2:1 (choloroform:TRIzol), samples were manually shaken and incubated at room temperature for 3 minutes. Samples were phase separated by centrifugation at 12,000×g at 4°C for 15 minutes, after which the aqueous layer was carefully isolated and mixed with an equal volume of 100% ethanol. RNA was further purified using an RNeasy Mini Kit (74106, QIAGEN) according to the manufacturer’s instructions (RNeasy Mini Handbook, 4th Ed., June 2012) with the following deviations: for each sample, a final 30 µL elution was performed twice, isolating 60 µL of RNA in total into each collection tube. An on-column DNase digestion step was also performed under the same instructions using an RNase-Free DNase Set (79254, QIAGEN). Final RNA concentration and sample purity were determined via a NanoDrop 1000 spectrophotometer (Thermo Fisher).
Once obtained, RNA samples were transferred on dry ice to the Georgia Genomics and Bioinformatics Core at UGA for library preparation and sequencing. There, IP samples were first concentrated using solid phase reversible immobilization (SPRI) beads. Then, the TruSeq Stranded Total RNA Library Prep Gold kit (20020598, Illumina) was used to deplete rRNA and prepare stranded cDNA libraries from all twenty-four samples. These uniquely barcoded cDNA libraries were then pooled by sample type, forming one IP library pool and one Input library pool. Each pool was sequenced on a separate NextSeq High Output Flow Cell (Illumina) for 150 cycles to generate paired-end, 75 base-pair (bp) reads.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
A fraction of initial head lysate volumes were isolated to generate Input samples; the remainder underwent IP protocols. Thus, PUT samples are derived from this isolate, 20% of the initial head lysate volumes.
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| Data processing |
Many of the data processing steps detailed below were conducted on the Galaxy web platform, specifically using the public server at usegalaxy.org. For each Galaxy tool described, exact parameters and version numbers used are also detailed in a Supplemental Table of the Rounds et al. submission associated with this accession.
To start on Galaxy, for each sample, reads from across all four NextSeq flow cell lanes were concatenated using the “Concatenate datasets tail-to-head” tool (Galaxy Version 1.0.0).
Reads were mapped using RNA STAR (Dobin et al. 2013), Galaxy Version 2.6.0b-1 and default parameters with the following exceptions: read type—paired (as individual datasets), reference genome—from history (using the BDGP6.22, Ensembl 97 FASTA and GTF), and length of the genomic sequence around annotated junctions—74.
Mapped reads were assigned to exons/genes and tallied using featureCounts (Liao et al. 2014), Galaxy Version 1.6.3+galaxy2 and default parameters with the following exceptions: specify strand information—Stranded(Reverse), gene annotation file—history (using the BDGP6.22, Ensembl 97 GTF), create gene-length file—true, count fragments instead of reads—enabled, GFF gene identifier—gene_name.
To enable inter-sample read count comparisons, count normalization and differential expression analysis was conducted using DESeq2 (Love et al. 2014), Galaxy Version 2.11.40.2 and default parameters with the following exceptions: factors—3 levels, each a dataset collection of four biological replicates, output normalized counts table—true, output all levels vs all levels—true. Importantly, DESeq2 analysis was performed twice, once on the 12 IP samples and once on the 12 Input samples; for discussion of this sample separation method see the Supplementary Materials and Methods of the Rounds et al. submission associated with this accession.
To identify Nab2-associated and Atx2-associated RNAs, we calculated IP/Input (i.e. “percent input” or “percent recovery”) (Zhao et al. 2010; Aguilo et al. 2015; Li et al. 2019) values for each of a “testable” set of 5,760 genes with 1)detectable expression in all twelve Inputs and 2)an average normalized Nab2 or Atx2 RIP read count greater than 10. These criteria were based on those used in (Lu et al. 2014; Malmevik et al. 2015). Fold enrichment differences were statistically tested by performing gene-by-gene one-way ANOVAs (Li et al. 2019), applying Dunnett’s post-hoc test (Dunnett 1955), and calculating adjusted p-values corrected for multiple hypothesis testing within each gene-by-gene ANOVA. We identify a small, focused set of statistically significantly enriched RNAs using this approach, arguing type I (i.e. false positive) error is sufficiently controlled and additional corrections between genes are not necessary (Rothman 1990). See Methods of the Rounds et al. submission associated with this accession for more detail.
Genome_build: BDGP6.22 (downloaded from release 97 of the Ensembl database)
Supplementary_files_format_and_content: The "Percent_Input_..." file is a tab-delimited text file containing results of RBP-RNA ASSOCIATION SIGNIFICANCE TESTING for all 5,760 genes in the TESTABLE SET (see data processing steps for set definition). The essential values to identify SIGNIFICANTLY RBP-ASSOCIATED TRANSCRIPTS are listed in the first 6 columns; these values are gene name, properly normalized % Input values per genotype, and the results of RBP-RNA association statistical significance testing. Later columns give further details or illustrate calculations used to arrive at the final results in the first 6 columns. Genes are ordered first by Nab2-FLAG fold enrichment ("% Input F Norm AVG"); then, all genes with a value in that field greater than 1 are ordered, smallest to largest, by adjusted Dunnet's post-hoc test p-value (FvsW DunAdjp). In all, for all testable genes, IP/Input (i.e. % input or fold enrichment) values are reported at both the level of overall genotype (e.g. elav>Nab2-FLAG or "F") and individual sample pair (e.g. elav>Nab2-FLAG RIP and PUT sample #2). This report also includes gene-level results of gene-by-gene one-way ANOVAs (for association significance testing) along with averages of DESeq2-normalized counts per genotype and RIP or PUT sample type combination (e.g. W.RIP). Where appropriate, standard deviation (SD) and standard error of the mean (SEM) values are also reported.
Supplementary_files_format_and_content: The DESeq2-normalized read counts file ("DESeq2_Normalized_Counts-combined...") is a tab-delimited text file containing a matrix of NORMALIZED READ COUNTS for all 24 samples. Normalized counts were generated by two independent DESeq2 analyses, one on the 12 RIP samples and one on the 12 PUT samples, and then combined into this matrix. See "data processing step" text for further detail. DESeq2 normalization accounts for library size and composition, enabling accurate comparison of relative RNA abundance for a given gene between samples. FPKM and similar methods do not account for library composition. Critically though, DESeq2-normalized counts should not be directly compared between different genes—instead, genes should be compared by comparing relative fold changes or enrichment values. The inclusion of "Parametric" in the file name refers to the "fit type" setting used when running DESeq2.
Supplementary_files_format_and_content: All "…[featureCounts…" files are .tabular files that contain, for a single sample, unedited, non-normalized RAW COUNTS per gene as assigned by featureCounts analysis of the appropriate .bam read mapping/alignment file produced by STAR. These files are may be opened by text editors and Microsoft Excel.
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| Submission date |
Jan 27, 2021 |
| Last update date |
Feb 05, 2021 |
| Contact name |
Ken Moberg |
| E-mail(s) |
[email protected]
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| Phone |
4042177708
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| Organization name |
Emory University School of Medicine
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| Department |
Cell Biology
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| Lab |
Room 442
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| Street address |
615 Michael Street
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| City |
Atlanta |
| State/province |
Georgia |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE165677 |
The disease-associated proteins Drosophila Nab2 and Ataxin-2 interact with shared RNAs and coregulate neuronal morphology |
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| Relations |
| BioSample |
SAMN17611053 |
| SRA |
SRX9964614 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5048085_Galaxy184-_featureCounts_on_W-PUT-2_Counts_.tabular.txt.gz |
78.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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