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| Status |
Public on Sep 16, 2016 |
| Title |
Scer_SSii/Mn_DMSvivo_Rep1_genomeWide |
| Sample type |
SRA |
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| Source name |
BY4741 cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
molecule subtype: mRNA
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| Treatment protocol |
For the in vivo DMS treatment of S. cerevisiae, DMS was added directly to the growing log phase yeast culture at 5% for 3-4 min. 25% BME, 30% isoamyl alcholol was used to quench the DMS. Cells were lysed with SDS, and RNA was isolated with hot acid phenol. For denatured samples, RNA was first extracted and then heated to 95deg for treatment with DMS.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using hot acid phenol. mRNA was randomly fragmented using Ambion fragmentation reagents, followed by PNK, and ligation using T4 K227Q truncated RNA ligase. rRNA was depleted post-ligation using RiboZero (Epicentre), and reverse transcription was done with Superscript II in Mn2+ buffer. Circ ligase (Epicentre) was used for circularization, and Illumina adapters were introduced by 8-10 cycles of PCR.
Cells or oocytes were treated with DMS for 5mins, total RNA was extracted with Triazol, rRNA was subtracted with Ribozero (yeast and mammalian cells) or RNaseH with specific oligoes (Drosophila)
Gene specific RT/PCR followed by Nextera XT or LMPCR. For genome-wide libraries we used 3' ligation , followed by circularixation, and PCR to add the sequencing adapters.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Illumina bcl2fastq v2.17 was used for base-calling.
Alignments were performed using Tophat v2.1.0 with bowtie2 and the following options for 50bp sequencing run: --no-novel-juncs -N 5 --read-gap-length 7 --read-edit-dist 7 --max-insertion-length 5 --max-deletion-length 5 -g 3
Non-uniquely mapping reads were removed. Additionally, 2nt or 5nt were removed from the 5' end of each read for TGIRT and SSii data, respectively, due non-template addition during reverse transcription.
Mismatch locations were called, and mismatches located within 3nt of an insertions or deletion were masked for future analysis.
In the case of data with a unique molecular index (UMI), sequencing reads were filtered for length >100nt and for unique reads only. For data generated with NexteraXT tagmentation, alignments to both strands were combined.
Genome_build: Saccharomyces cerevisiae assembly R64 (UCSC: sacCer3) or, for human data, the longest RefSeq isoforms from hg19.
Supplementary_files_format_and_content: Tab-delimited files have three columns, representing the name of the aligned sequenced (either chromosome or transcript name), the position within that sequence, and the ratiometric DMS-MaPseq value calculated as the number of mismatches divided by the total sequencing depth at each postion. The position number directly indicates the DMS modified position starting at 1.
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| Submission date |
Jul 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Silvi Rouskin |
| E-mail(s) |
srouskin@wi.mit.edu
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| Organization name |
Whitehead Institute
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| Street address |
455 Main Street
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02141 |
| Country |
USA |
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| Platform ID |
GPL21656 |
| Series (1) |
| GSE84537 |
DMS-MaPseq: A genome-wide or targeted approach for RNA structure probing in vivo |
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| Relations |
| BioSample |
SAMN05414722 |
| SRA |
SRX1959212 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2241647_SSii_DMSvivo_Yeast_rRNA_COUNTS_Minus_NormSignal_Rep1.txt.gz |
63.7 Kb |
(ftp)(http) |
TXT |
| GSM2241647_SSii_DMSvivo_YeastmRNA_COUNTS_Minus_NormSignal_Rep1.txt.gz |
11.2 Mb |
(ftp)(http) |
TXT |
| GSM2241647_SSii_DMSvivo_YeastmRNA_COUNTS_Plus_NormSignal_Rep1.txt.gz |
11.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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