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Status |
Public on Jul 23, 2021 |
Title |
2days_rep1_ssRNase_noprotein |
Sample type |
SRA |
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Source name |
MEL cells 2 days post differentiation, ssRNase treated, without proteinase K
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Organism |
Mus musculus |
Characteristics |
cell line: MEL cells differentiation stage: 2 days
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Treatment protocol |
Undifferentiated and differentiated samples were treated with formaldehyde to induce crosslinking of RNA binding proteins to RNA molecules.
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Growth protocol |
MEL cells were grown under standard conditions in minimal essential medium (MEM) and supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1x antibiotic antimycotic (Invitrogen). MEL cells in suspension culture at the log phase of growth at a density of 2 x 105/ml were supplemented with 2% DMSO (Sigma) to induce differentiation, and cells were collected at various time points for further analysis.
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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).
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Library strategy: PIP-seq 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 |
Dec 17, 2019 |
Last update date |
Jul 23, 2021 |
Contact name |
Mengge Shan |
E-mail(s) |
mengge@pennmedicine.upenn.edu
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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 |
GPL17021 |
Series (1) |
GSE142242 |
Global Analysis of the RNA-Protein Interaction and RNA Secondary Structure Landscapes Identifies Dynamic Changes During Mammalian Erythropoiesis |
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Relations |
BioSample |
SAMN13617388 |
SRA |
SRX7401891 |
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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