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Sample GSM1090053

Query DataSets for GSM1090053

Status

Public on Nov 06, 2014

Title

Wt_rep2 [do1556_PolIII_skin_1900_mmudo1411do1411_wt_CRI01]

Sample type

SRA

Source name

total mouse skin

Organism

Mus musculus

Characteristics

background strain: C57Bl6/J CBA F1
genotype/variation: WT
antibody: antibodies recognizing antigen POLR3A, the RPC1/155 subunit of Pol III as indicated in (Kutter, Brown et al. 2011)

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 fixed in 1% formaldehyde (v/v).

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.

Extracted molecule

genomic DNA

Extraction protocol

Fixed material was lysed, sonicated and then incubated with antibodies recognizing antigen POLR3A, the RPC1/155 subunit of Pol III as indicated in (Kutter, Brown et al. 2011). Input material was lysed, sonicated and but not incubated with antibodies.
The immunoprecipitated and input material was end-repaired, A-tailed, ligated to the sequencing adapters, amplified by 18 cycles of PCR and size-selected (200-300 base pairs). Each experiment was done on skin material isolated from 3 3.5-weeks old male mice pooled together. At least two independent biological replicates from wild type and one for NSun2 -/- animals were carried out.
Pol III ChIP sequencing was performed from back skin from two wild-type independent biological replicates and one NSun2 -/- independent biological replicate. Input libraries were only from one wild-type independent biological replicate and one NSun2 -/- independent biological replicate. Each biological replicate were done from material from 3 mice pooled together. 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).

Library strategy

ChIP-Seq

Library source

transcriptomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer II

Description

polIII.counts.dat

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

Submission date

Feb 28, 2013

Last update date

May 15, 2019

Contact name

Patrick Lombard

E-mail(s)

pdl30@cam.ac.uk

Organization name

University of Cambridge

Street address

Tennis Court Road, Cambridge