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| Status |
Public on Jun 15, 2022 |
| Title |
60min_rep1_ssRNase_plusP |
| Sample type |
SRA |
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| Source name |
P. patens protonema
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| Organism |
Physcomitrium patens |
| Characteristics |
differentiation stage: 60 min ABA
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| Treatment protocol |
10 -day old P. patens protonema tissue was sonicated and then transferred to 250 mL liquid PpNH4 media. After 3 days of growth on a plate shaker at ~100 rpm, ABA was added to the liquid culture for a final concentration of 10 mM and the same volume of methanol was added to the control samples. Samples were placed back on the shaking incubator for 30 or 60 minutes before being collected and flash frozen with liquid nitrogen.
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| Growth protocol |
Physcomitrella patens was grown on PpNH4 media plates for 7-10 days and then propagated following instructions provided by the Benzanilla lab. Specifically, samples are homogenized until no big clumps remain visible. Each new plate is layered with a sterile cellophane and 0.5 mL of the tissue mixture is placed on the cellophane. The plate is then gently swirled to evenly distribute the tissue and sealed with micropore plate. Then the plates are placed in Percival chambers at 25C with 16 hours/8 hours light/dark cycles.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Homogenized samples were divided into 2 subsamples one for interrogation of protein binding, the other for interrogation of secondary structure. For protein binding samples, each subsample were then was further divided and half was incubated in either 100 U/ml of a single-stranded RNase (ssRNase) (RNaseONE (Promega; Madison, WI, USA)) with 200 μg/ml BSA in 1X RNaseONE buffer for 1 hour at room temperature (RT), or 2.5 U/ml of a double-stranded RNase (dsRNase) (RNaseV1 (Ambion; Austin, TX, USA)) 1X RNA structure buffer for 1 hour at 37°C as previously described (Anderson et al., 2016). Following RNAse treatment, proteins were denatured and digested by treatment with 1% SDS and 0.1 mg/ml Proteinase K (Roche; Basel, Switzerland) for 15 minutes at RT. Finally, RNAse crosslinks were reversed by incubation at 65°C for 2 hours. For structure only samples, we performed the identical treat-ments as described for protein bound samples, except that the cross-linked homogenate was treated with 1% SDS and 0.1 mg/ml Proteinase K (Roche;Basel,Switzerland)and ethanol precipitated before treatment with the structure specific RNAses. Post digestion, RNA was then isolated using Qiagen miRNeasy RNA isolation kit as described in the manual.
Strand specific libraries were constructed as previously described (Silverman et al, 2014).
PIP-seq
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
P. patens protonema with 60 minutes ABA, ssRNase treated, protein kinase treated
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| Data processing |
PIP-seq reads were trimmed using cutadapt to remove 3’ sequencing adapters (cutadapt version 1.2.1 with parameters –e 0.006 –O 6 –m 14). Resulting trimmed reads were then collapsed into unique reads and aligned to the mm10 mouse genome sequence using TopHat (version 2.0.10 with parameters – library-type fr-secondstrand –read-mismatches 2 –read-edit-dist 2 –max-multihits 10 –b2-very-sensitive –transcriptome-max-hits 10 –no-coverage-search –no-novel juncs). Any PCR duplicates were collapsed to single reads for all downstream analysis.
PPSs were identified using a modified version of the R package CSAR (Muino et al., 2011). Read coverage values were calculated for each base in the genome and a Poisson test was used to determine an enrichment score for footprint as compared to structure only libraries. PPSs were then called with a false discovery rate of 5% as previously described (Silverman et al., 2014).
Structure scores for each transcript are based on the coverage for dsRNA-seq and ssRNA-seq coverages for each base in detectable transcripts from structure-only samples in each replicate. The structure score for each base was calculated as previously described (Gosai et al., 2015; Silverman et al., 2014).
Genome_build: mm10
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| Submission date |
Apr 09, 2020 |
| Last update date |
Jun 15, 2022 |
| Contact name |
Mengge Shan |
| E-mail(s) |
[email protected]
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| Organization name |
University of Pennylsvania
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| Department |
Biology
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| Lab |
Brian Gregory
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| Street address |
433 South University Avenue
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL17793 |
| Series (1) |
| GSE148423 |
Global analysis reveals large scale dynamics of protein-RNA interaction and RNA secondary structure characteristics of Physcomitrella patens ABA response (PIP-Seq) |
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| Relations |
| BioSample |
SAMN14569717 |
| SRA |
SRX8092731 |
| 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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