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| Status |
Public on May 02, 2024 |
| Title |
EpiKO_ZT12_4 |
| Sample type |
SRA |
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| Source name |
Skin
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| Organism |
Mus musculus |
| Characteristics |
tissue: Skin
cell type: epidermal cell
time point: ZT12
strain: C57BL/6
Sex: Male
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| 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).
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| 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.
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| 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 |
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| 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).
To identify specific genes reliant upon different clock communication pathways for their rhythmic expression, the DryR algorithm was applied to compare this dataset with rhythmic in WT epidermis. Raw RNAseq counts were used as input, and the function ‘dryseq()’ was applied to run the algorithm. The resulting output was then filtered (BICW ≥ 0.9, Amp ≥ 0.25, Periodicity = 24 hours), to generate the gene sets then further analyzed.
Assembly: mm10
Supplementary files format and content: Text file (.txt) output from the DryR algorithm when applied to the diurnal RNA-seq data sets presented here and that of WT epidermis (as presented in GSE190032) . The file contains the probability that each gene is associated with a specific rhythmic gene model, along with other details on the rhythmicity parametres of the gene.
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| Submission date |
Feb 09, 2024 |
| Last update date |
May 02, 2024 |
| Contact name |
Thomas Mortimer |
| E-mail(s) |
thomas.mortimer@irbbarcelona.org
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| Organization name |
IRB Barcelona
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| Street address |
Carrer Baldiri Reixac 10-12
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| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE190035 |
Characterisation of the epidermal diurnal and circadian transcriptomes of mice with tissue-specific circadian clock activity |
| GSE255474 |
Characterisation of the epidermal diurnal transcriptome of mice with K14-Cre-driven Bmal1 knockout |
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| Relations |
| BioSample |
SAMN39903273 |
| SRA |
SRX23585490 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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