|
| Status |
Public on May 10, 2024 |
| Title |
Rat_9_con_distal |
| Sample type |
SRA |
| |
|
| Source name |
distal colon
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Wistar Munich
tissue: distal colon
treatment: control
gender: male
|
| Treatment protocol |
Animals were housed at 22°C and with a 12:12 h light:dark cycle. During the experiment, the rats were housed in single-animal metabolic cages (Techniplast), where animal weight, water and food consumption as well as urine and faeces output were monitored daily.
For an initial three days, the rats were acclimatized to the metabolic cages with ad libitum access to water and standard rodent chow. Subsequently, six rats were switched to standard rodent chow mixed with 1% w/w sodium cholate (Sigma Aldrich).
|
| Growth protocol |
Twelve male Wistar Munich rats were housed in standard cages with ad libitum access to water and standard rodent chow (cat. no. 1321, Altromin). Animals weighed between 284g and 347g, when they were put in metabolic cages.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 10 days on the different diets rats were euthanized by cervical dislocation, their abdomens opened and the colons were dissected out and flushed clean off faeces with cold PBS.
Colons were divided into thirds and the middle section discarded. The proximal segment (initial 1/3) and the distal segment (last 1/3) were inverted, tied together at both ends and filled with Ca2+ free Ringer buffer with EDTA to form small sacs.
The colon sacs were put in separate 50 mL tubes with warm Ca2+ free Ringer buffer with EDTA and incubated at 37°C for 20 min. The tubes were shaken vigorously to release colonic epithelial cells (CECs) from the colon sacs. Colon sacs were then removed and the CEC suspensions were divided into two tubes and centrifuged at 4000 g for 3 min to pellet the cells. Supernatants were discarded and pellets were resuspended and homogenized in either 1 mL TriReagent™ (Ambion®) for RNA-analysis or in dissection buffer for protein analysis.
TriReagent samples were sonicated and RNA was extracted using the RiboPure™ RNA Purification kit (Ambion®) according to the manufacturer’s instructions. RNA yield was estimated based on A260 measurements by spectrophotometry using a NanoPhotometer® (Implen) with Elution Buffer from the RiboPure™ kit as blank.
Preparation of strand-specific cDNA libraries, RNA selection using polyA purification and single channel RNA sequencing was performed by Eurofins Genomics (Germany) using HiSeq SBS Kit v4 on a HiSeq 2500 (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
HiSeq Control Software 2.2.58 RTA 1.18.64 bcl2fastq-1.8.4 software was used for basecalling.
Sequences are demultiplexed according to the 2x8 bp index code with 1 mismatch allowed. All reads are passed filter, i.e. reads have passed the default Illumina filter procedure (chastity filter).
BWA-MEM version 0.7.12-r1039 was used to map RNA reads to reference sequences (Li H. (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv:1303.3997v2[q-bio.GN]).
Normalization of reads was performed using Trimmed Mean of M values (TMM) normalization method with the edgeR-package version 3.16.2 in R.
Genome_build: rn6
Supplementary_files_format_and_content: GEN160929_M.profiling-table.txt contains a Matrix table with both the raw counts and CPM normalized counts for every gene and every sample
|
| |
|
| Submission date |
May 11, 2021 |
| Last update date |
May 10, 2024 |
| Contact name |
Hanne B Moeller |
| E-mail(s) |
hbmo@biomed.au.dk
|
| Organization name |
Aarhus University
|
| Department |
Dept. of Biomedicine
|
| Street address |
Wilhelm Meyers Alle 3, building 1233
|
| City |
Aarhus |
| ZIP/Postal code |
8000 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL18694 |
| Series (1) |
| GSE174249 |
A systems-level analysis of bile acids effects on rat colon epithelial cells |
|
| Relations |
| BioSample |
SAMN19107669 |
| SRA |
SRX10855763 |
