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| Status |
Public on Nov 06, 2014 |
| Title |
Wt_rep2 [do1662_unk_skin_unk_mmuWT#2_CRI01] |
| Sample type |
SRA |
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| Source name |
total mouse skin
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| Organism |
Mus musculus |
| Characteristics |
background strain: C57Bl6/J CBA F1
genotype/variation: WT
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| Treatment protocol |
Mouse back skin was collected at P25 (postnatal day 25), when hair follicles start the growing phase or anagen phase of the hair cycle. Skin was shaved before collection, and immediately snap-frozen in liquid N2.
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| Growth protocol |
Total mouse back skin from wild-type and NSun2 -/- males in anagen (postnatal day 25) were used. NSun2 -/- mice (GGTC-clone ID: D014D11) were generated by the German Gene Trap Consortium, and bred under a C57Bl6/J CBA F1 background. Wild-type mice were coming from the same breedings as NSun2 -/- mice.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from mouse skin was prepared using Trizol reagent (Invitrogen) according to the manufacturer's instructions and then treated with DNase (Turbo DNase, Ambion).
Total RNA was extracted as described previously, DNase and Ribo-zero treated. The remaining RNA fraction was bisulfite-converted as followed; about 2 μg of RNA was mixed in 70μl of 40% sodium bisulfite pH 5.0 and DNA protection buffer (EpiTect Bisulfite Kit, Qiagen). The reaction mixture was incubated for four cycles of 5 min at 70ºC followed by 1 hour at 60ºC and then desalted with Micro Bio-spin 6 chromatography columns (Bio-Rad). RNA was desulfonated by adding an equal volume of 1M Tris (pH 9.0) to the reaction mixture and incubated for 1h at 37°C, followed by ethanol precipitation.The bisulfite-converted RNA quality and concentration was then assessed on a Bioanalyzer 2100 RNA nano-chip (Agilent). About 120 ng of bisulfite-converted RNA was used to generate Bisulfite-seq libraries. Bs-Seq libraries were performed according to TruSeq Small RNA preparation kit (Illumina). Since the sample was already sufficiently fragmented by the bisulfite-conversion reaction (about 50-120nt), the size selection step prior to adaptor hybridization and ligation was not performed. First 2’,3’-cyclic phosphate and 5’-hydroxyl termini produced during the bisulfite/desulfonation reaction were end-repaired with T4 PNK and Spermidine (New England Biolabs). RNA adapters suitable for Illumina sequencing were then ligated, reverse-transcribed at 50ºC for 1 hour with SuperScript III and 1mM of each dNTP (SuperScript III cDNA synthesis kit, Invitrogen) followed by 18 cycle-PCR amplification. The amplified product was not size-selected before sequencing.
RNA bisulfite sequencing of RNA from back skin from four wild-type independent biological replicates and four NSun2 -/- independent biological replicates were used. All libraries were sequenced on the Illumina Genome Analyzer II (single-ended and 100 pb read length) and post-processed using the standard GA pipeline software v1.4 (Illumina).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
RNA Methylation profiling by high throughput sequencing
Reads were aligned to the mouse reference genome (GRCm38/mm9) using Bismark (version 0.7.6). Only the reads uniquely aligning to the genome and aligning without mismatch were considered for further analysis.
small non-coding RNA profiling by high throughput sequencing
Adapters were removed, and the 20-200 nt reads were aligned to the mouse and human reference genomes (UCSC mm9 and GRCh37/hg19) with options m 500 -v2 -best -strataas decribed for PolII ChIP-seq reads. To account for the addition of CCA 3'-ends to mature tRNAs, unaligned reads were trimmed of CCA[CCA] ends and realigned using the same options. Annotations were conducted based on the gene loci predicted by tRNAscan-SE in the mouse and based on predicted tRNA genes from GtRNAdB (http://lowelab.ucsc.edu/GtRNAdb) in human. Fragments that matched at multiple tRNA genes were distributed proportionally to the fraction of uniquely matching fragments at these regions as described (Kutter et al. 2011/2?). Reads with CCA[CCA] ends that covered at least 90% of the tRNA gene were considered mature tRNAs. For fragment aligning at tRNA genes, reads that exceeded the gene start or end by more than 10 % were discarded. For all mature tRNAs and fragments aligning at tRNA genes, counts were normalized and differential abundance of the fragments was evaluated with the Bioconductor/R package DESeq (Anders and Huber, 2010).
Pol III Chromatin-IP profiling by high throughput sequencing
In the mouse reference genome (UCSC NCBI37/mm9), 3,282 candidate tRNA genes were predicted with tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE) including genes with an 'undetermined' isoacceptor family; loci marked as pseudogenes were discarded. PolIII ChIP-seq reads were aligned to the reference genome using Bowtie (Langmead et al, 2009). Two mismatches and at most 500 multiple matches were tolerated, i.e. specifically the Bowtie options m 500 -v2 -best -strata
Genome_build: mm9
Supplementary_files_format_and_content: GSM1090034..GSM1090041: Tabdeliminated text file methylation report. Columns contain chromosome, start, end, percentage methylation, methylated calls, unmethylated calls.
Supplementary_files_format_and_content: GSM1090042..GSM1090049: Tab deliminated text file containing counts for the tRNA's
Supplementary_files_format_and_content: GSM1090050..GSM1090054: Tab deliminated text file containing counts for the tRNA's
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| Submission date |
Feb 28, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Patrick Lombard |
| E-mail(s) |
pdl30@cam.ac.uk
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| Organization name |
University of Cambridge
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| Street address |
Tennis Court Road, Cambridge
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| City |
Cambridge |
| State/province |
Cambridgeshire |
| ZIP/Postal code |
CB21QR |
| Country |
United Kingdom |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE44746 |
Loss of cytosine-5 methylation in tRNA triggers stress responses in a disease model for Intellectual Disability |
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| Relations |
| SRA |
SRX248377 |
| BioSample |
SAMN01974676 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1090035_do1662.results.txt.gz |
1.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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