NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM4470205 Query DataSets for GSM4470205
Status Public on Jun 15, 2022
Title 00min_rep2_dsRNase_plusP
Sample type SRA
 
Source name P. patens protonema
Organism Physcomitrium patens
Characteristics differentiation stage: 00 min ABA
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.
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.
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
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2500
 
Description P. patens protonema with 00 minutes ABA, dsRNAse treated, proteinase K treated
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
 
Submission date Apr 09, 2020
Last update date Jun 15, 2022
Contact name Mengge Shan
E-mail(s) [email protected]
Organization name University of Pennylsvania
Department Biology
Lab Brian Gregory
Street address 433 South University Avenue
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
 
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)
Relations
BioSample SAMN14569730
SRA SRX8092719

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap