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| Status |
Public on Jul 28, 2022 |
| Title |
DMS_pladB_Rep1 |
| Sample type |
SRA |
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| Source name |
YOH001
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: YOH001
treatment: +pladB, +DMS
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| Treatment protocol |
We carried out splicing inhibition and DMS treatment for two biological replicates, splicing inhibition only for a no-modification control, and DMS treatment only for the control without splicing inhibition. For conditions with splicing inhibition, we treated cultures with 5 μM pladB using 50 μL of 1 mM pladB for every 10 mL of culture, and we incubated cultures at 30 oC for 1 hour with shaking. We treated cultures with 3% DMS or an equivalent volume of H2O for the no-modification control. Treated cells were incubated at 30 °C for 5 minutes, and the reaction was quenched with 20 mL stop solution (30% 2-mercaptoethanol, 50% isoamyl alcohol) for every 10 mL of culture.
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| Growth protocol |
Strains were grown at 30 oC on YPD plates and in YPD liquid medium. Single colonies of YOH001 were used to inoculate overnight cultures, diluted to OD600 0.1, and grown to OD600 0.5-0.6. Biological replicates were obtained from distinct single colonies.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the YeaStar RNA Kit (Zymo Research), using 7.5 μL of Zymolase for every 2.5 mL starting cell culture and shortening the Zymolase incubation to 15 minutes at 30 oC to reduce RNA fragmentation.
We first depleted the extracted RNA of rRNA using RNase H to deplete rRNA with complementary oligos. We used an RNA Clean and Concentrator-5 (Zymo) column with the size-selection protocol to exclude RNA below a size cutoff of 200 nucleotides. RNA was fragmented with Ambion fragmentation reagents, end-repaired with rSAP (NEB), and then ligated to an adenylated universal DNA cloning linker with T4 RNA ligase 2 truncated KQ (NEB). Excess DNA linker was degraded with 5’ Deadenylase (NEB) and RecJf (NEB). We reverse transcribed RNA with TGIRT enzyme (InGex), and we size-selected cDNA (200 - 400 nucleotide size range) using a denaturing PAGE gel extraction to remove excess RT primer. cDNA was circularized with CircLigase ssDNA Ligase (Lucigen), and index sequences and Illumina sequencing adapters were added via PCR to generate the final library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
d1_d2_combined_rfcount_rfnorm_processed.tar.gz
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| Data processing |
cutadapt was used remove adapter sequences including indexing primer sequences and the universal cloning linker.
Sequencing reads were aligned to various sequence sets of interest, including introns, complete pre-mRNA ORFs for genes containing introns, and coding mRNA sequences for genes containing introns, rRNA sequences, and structured control RNAs. Paired-end alignment was performed with Bowtie2 using the alignment parameters: --local --sensitive-local --maxins=800 --ignore-quals --no-unal --mp 3,1 --rdg 5,1 --rfg 5,1 --dpad 30. For analysis combining replicates, alignment files were merged with samtools. Alignments were sorted and indexed using samtools.
UMI-tools was used to extract UMI tags from reads prior to removing adapter sequences by matching to the expected pattern from our RT primers, and after alignment, reads with matching mapped positions and UMI tags were then deduplicated using the UMI-tools60 dedup function with default parameters for paired-end reads.
Mutational frequencies and coverage values were obtained by processing alignment files using RNAframework executables. For each library and reference sequence set, first rf-count was run with the flag -m to compute mutation counts, using all other default parameters. Coverage statistics were obtained for each sequence with rf-rctools stats, and per-position mutation counts and per-position coverage statistics were obtained with rf-rctools view.
Normalized reactivity values were obtained with rf-norm, using Rouskin scoring with 90% Winsorizing for normalization, reactive bases A and C, and dynamic resizing of the normalization window to account for A/C frequencies (flags: -sm 2 -nm 2 -ow -rb AC -dw).
Assembly: S. cerevisiae intron reference sequences were obtained from Talkish, et al. 2019 (doi: 10.1371/journal.pgen.1008249). Coding ORF annotations were obtained from the Saccharomyces Genome Database for the S288C reference genome. Pre-mRNA reference sequences were obtained using the sacCer3 UCSC genome assembly.
Supplementary files format and content: Coverage values, mutational frequencies, and reactivity values are provided for all introns. RNA count files (.rc) and index files (index.rci) from the rfcount module of RNAframework are included. Also provided are coverage values from from rf-rctools stats (files ending with stats.txt), and per-position coverage and mutational frequency statistics (files ending with view.txt). Normalized reactivity values are included from the rfnorm module of RNAframework (one file per intron in folder ending with _reactivity/).
Supplementary files format and content: For biological replicates, combined processed data files are provided which include mutational frequencies, coverage, and reactivity values when combining reads from both replicates.
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| Submission date |
Jul 27, 2022 |
| Last update date |
Jul 30, 2022 |
| Contact name |
Rhiju Das |
| E-mail(s) |
rhiju@stanford.edu
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| Organization name |
Stanford University
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| Street address |
279 Campus Dr, B419 Beckman Center
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| City |
PALO ALTO |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL21656 |
| Series (1) |
| GSE209857 |
RNA structure landscape of S. cerevisiae introns |
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| Relations |
| BioSample |
SAMN29990596 |
| SRA |
SRX16688939 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6402544_d1_rfcount_rfnorm_processed.tar.gz |
640.4 Kb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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