|
| Status |
Public on May 02, 2024 |
| Title |
WT_ZT20_2 |
| Sample type |
SRA |
| |
|
| Source name |
Skin epidermis
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
replicate: 2
time point: ZT20
gender: Male
genotype: Arntl wt/wt ; K14Cre wt/tg ; Syt10-Cre tg/wt
clock reconsitution status: Wild-type (WT)
|
| Treatment protocol |
To profile daily transcriptional rhythms, for each genotype, the skin of four eight-week-old mice (all males) was collected at six time points throughout the day. Each time point was separated by four hours, with the first sample collected at lights on (ZT0/8:00am).
|
| Growth protocol |
Mice were kept in rooms at 22°C degrees with ad libitum access to a standard chow diet, under a 12h:12h light/dark cycle.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Mice were sacrificed at the relevant time by C02 asphyxiation, and their backskin was subsequently removed and stored in ice-cold PBS. The hypodermis was then removed from the skin by vigorous scraping using a scalpel. In a petri dish, the skin was floated on 10ml of 1mg/ml dispase (Sigma Aldrich) (in PBS) for 45 minutes at 37°C to enzymatically separate the epidermis. Using a scalpel, the epidermis was separated from the surface of the skin, and then mechanically disaggregated using surgical scissors. The disaggregated epidermis was placed into 10ml of ice cold PBS + 10% chelated FBS to inactivate dispase, and then sequentially filtered through 100µM and 40µM filters, giving a purified extract of epidermal cells. To extract total RNA, one million epidermal cells were lysed in 500µL TRIzol (Invitrogen), vortexed with 100µL chloroform, and then the resulting aqueous layer containing RNA was isolated. The aqueous layer was mixed with 825µL RLT buffer (Qiagen) and 625µL 100% ethanol to induce RNA precipitation. The sample was further processed using an RNeasy mini kit (Qiagen) in accordance with the manufacturers protocol, and RNA was eluted in molecular biology grade water.
Prior to library preparation, RNA integrity was estimated using an RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent). Libraries were prepared with KAPA Stranded mRNA-Seq Illumina® Platforms Kit (Roche) following the manufacturer´s recommendations. The libraries were sequenced on NovaSeq 6000 (Illumina) with a read length of 1x51bp+17bp+8bp using, following the manufacturer’s protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 3.4.4).
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
JTK_WT.RNAseq_LD.txt
BIOCYCLE_WT_LD.tsv
|
| Data processing |
Quality control and adapter trimming was performed using Trimmomatic (version 0.36).
Reads were aligned to the mm10 genome (UCSC) using the HISAT2 aligner (version 2.2.1), and subsequently sorted using SAMtools (version 1.3.1). After alignment, read quantification was performed using featureCounts (version 1.6.0) and the resulting count data was then normalized to generate RPKM values using the edgeR ‘rpkm’ function (version 3.18.1).
Genes exhibiting daily rhythms in expression were identified using the Jonckheere-Terpstra-Kendall (JTK_CYCLE) and BIOCYCLE algorithms. RPKM normalised gene expression values (Log2 transformed) were provided as input. A differential rhythmicity analysis was performed between all four samples using the algorithm DryR.
Genome_build: mm10
Supplementary_files_format_and_content: Text files (.txt) contain the output from the JTK_CYCLE and BIOCYCLE algorithm when applied to the diurnal RNA-seq data sets generated for each clock reconstitution condition. Files contain the rhythmic parameters calculated by the algorithm for each gene detected by RNA-seq. The dryR results file contains the raw output from the algorithm DryR when it is run to compare the rhythmicity of all four clock reconstitution conditions characterised.
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| |
|
| Submission date |
Dec 02, 2021 |
| Last update date |
May 02, 2024 |
| Contact name |
Thomas Mortimer |
| E-mail(s) |
thomas.mortimer@irbbarcelona.org
|
| Organization name |
IRB Barcelona
|
| Street address |
Carrer Baldiri Reixac 10-12
|
| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE190032 |
Characterisation of the epidermal diurnal transcriptome of mice with tissue-specific circadian clock activity [LD] |
| GSE190035 |
Characterisation of the epidermal diurnal and circadian transcriptomes of mice with tissue-specific circadian clock activity |
|
| Relations |
| BioSample |
SAMN23569696 |
| SRA |
SRX13486916 |
