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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 30, 2021 |
| Title |
Ribo_MsLV_M_Expr3 |
| Sample type |
SRA |
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| Source name |
liver
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype: Idua-W401X KI/KI
treat: rAAV9.2xsup-tRNATyr
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| Treatment protocol |
Patient fibroblasts were infected with lentiviral vectors in the presence of 8 µg/mL polybrene. G418 was added to culture media to a final concentration of 0.1 mg/mL or 0.5 mg/mL as indicated 24 hours prior to harvest. 6-week old MPS1 KI male mice were injected with rAA9.2xsup-tRNATyr by IV. 10 weeks post injection, mice were transcardially perfused with ice-cold PBS, and tissues were immediately dissected, snap-frozen in liquid nitrogen, and stored under -80 °C.
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| Growth protocol |
Patient and control fibroblasts were purchased from Coriell Institute and were maintained in EMEM supplemented with 15% (v/v) fetal bovine serum and antibiotics at 37 °C with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
PFB cells and ground mouse livers powder were lysed on ice in the presence of cycloheximide. Lysates were clarified by centrifugation. For ribosome profiling, monosomes were generated by RNaseI digest followed by ultracentrifugation and purified by miRNeasy mini kit.
Ribosome proteced RNA fragments ranging from 26-34 nt were gel purified. Upon dephosphorylation of sample RNA, linker was ligated and this product was followed by reverse transcription and gel purification. Then the RT product was circularized by CircLigase ssDNA ligase followed by rRNA depletion with biotinylated subtraction oligo pool. Libraries were then PCR-amplified, and gel-purified libraries were pooled in equimolar ratios and submitted for sequencing.
Ribosome profiling
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ribosome proteced RNA fragments
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| Data processing |
Illumina bcl2fastq was used to convert base call (BCL) files into FASTQ files.
3′ adapter sequences were removed using Trimmomatic (with parameters ILLUMINACLIP, minlength, 10; seed mismatches, 2; palindrome clip threshold, 30; simple clip threshold, 5). Then 1 nucleotide from the 3' end of each read was trimmed using FASTX-Toolkit Trimmer.
The trimed reads that mapped to noncoding RNA sequences (rRNA, from RefSeq; Mt_rRNA, Mt_tRNA, rRNA, miRNA, scRNA, scaRNA, snoRNA, snRNA, sRNA, vaultRNA from GENCODE v30 were discarded. STAR8 was used for this alignment step with following parameters: --limitBAMsortRAM 20000000000 --outFilterMismatchNmax 1 --outFilterMultimapNmax 100 --alignIntronMax 1 --outWigType wiggle read1_5p.
The remaining reads were mapped to the hg38 human or mm10 mouse genome using annotations from GENCODE (v30) and (v25), respectively. For this step, STAR was used with following parameters to keep only uniquely mapped reads: --alignSJDBoverhangMin 1 --alignSJoverhangMin 8 --outFilterType BySJout --outFilterMultimapNmax 200 --outSAMmultNmax 1 --outMultimapperOrder Random --outWigType wiggle read1_5p.
Following alignment, reads were assigned to a custom transcriptome annotation where single transcripts are selected for each protein-coding gene. Further data analysis was performed on the Ribosome-Profiling pipeline of the DolphinNext, available at https://dolphinnext.umassmed.edu/index.php?np=1&id=695.
Genome_build: hg38 human or mm10 mouse
Supplementary_files_format_and_content: csv files containing raw counts and FPKM values for each Sample
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| Submission date |
Jul 01, 2021 |
| Last update date |
Nov 30, 2021 |
| Contact name |
Yue Zhang |
| E-mail(s) |
6leensky@gmail.com
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| Organization name |
Umass medical school
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| Street address |
GeneTherapyCenter, 368 Plantation st
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| City |
Worcester |
| State/province |
Massachusetts |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE179274 |
AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice (ribosome profiling) |
| GSE179275 |
AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice |
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| Relations |
| BioSample |
SAMN19989666 |
| SRA |
SRX11326844 |
| 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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