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| Status |
Public on May 04, 2019 |
| Title |
SHR 2 epithelium RRBS |
| Sample type |
SRA |
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| Source name |
proximal colonic epithelium
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| Organism |
Rattus rattus |
| Characteristics |
tissue: colon
cell type: SHR epithelium
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| Growth protocol |
Colonic epithelia were isolated as previously described. Briefly, freshly obtained proximal colon tissue was sequentially incubated with: (1) HBSS (VWR, Radnor, PA) containing 1.5mM DTT and 3mM EDTA on ice for 20 min; (2) HBSS containing 1.5mM EDTA at 37oC for 10 min. Then the tissue was shaken for 3 min to separate the epithelium from the remaining tissue. The epithelium suspension was centrifuged at 1500 rpm at 4oC and washed with HBSS containing 10% FBS. The cells obtained were aliquoted and stored at -80oC until RNA was extracted.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from colonic epithelia of WKY and SHR using the Quick DNA Miniprep Kit (Zymo Research, Irvine, CA).
RRBS libraries were constructed using the Diagenode premium RRBS kit C02030032 (Diagenode Inc. USA, Denville, NJ). Briefly, 100ng of genomic DNA was digested with Msp I enzyme at 37oC for 12 hours. End preparation (End repair and A-tailing) was conducted for TA-adaptor ligation in the following step. After ligation, the mixture was incubated with AMPure XP Beads (Beckman Coulter, Brea, CA) at room temperature for 15 min to allow DNA binding to the beads. The DNA bound to beads was washed prior to resuspension in buffer. After quantification of the resuspended DNA sample by real time PCR, equal amounts of each sample were pooled. The pooled library was treated with bisulfite and purified using columns according to the manufacturer’s protocol. Finally, the library was amplified by real time PCR and purified with AMPure XP beads.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Obtained RRBS data were analyzed using Qiagen CLC Main Workbench V12 (Qiagen, Germantown, MD) with Bisulfite Sequencing Plugin according to manufacturer’s instruction. Critical parameters were set as below: (1) mapping options: match score 1; mismatch cost 2; linear gap cost: insertion 3 and deletion 3; length faction 0.5; similarity fraction 0.8; reads with equal mapping score were assigned randomly; enable automatic paired distance estimation. (2) Sample thresholds: minimum high confidence site coverage 1; minimum high site count 1; maximum mean site coverage 0.0. Specific sites were analyzed using Fisher exact test. (3) The first 5 bases on the 5’-end of each read were excluded from methylation analysis, due to the common technical bias introduced by end-repair steps in RRBS library preparation. (4) The reference genome of Rattus Norvegicus (ensembl_v91) was obtained from CLC benchwork 12 reference data library function. The CpG sites with more than 20 sequencing reads were included in the analysis.
Supplementary_files_format_and_content: clc
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| Submission date |
May 03, 2019 |
| Last update date |
May 04, 2019 |
| Contact name |
tao yang |
| E-mail(s) |
taoyang0923@gmail.com
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| Organization name |
University of Florida
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| Street address |
McKnight Brain Institute, 1149 Newell Drive
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| City |
Gainesville |
| State/province |
Florida |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platform ID |
GPL26616 |
| Series (2) |
| GSE130669 |
RRBS of colonic epithelium of WKY and SHR |
| GSE130671 |
Colonic epithelium of WKY and SHR |
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| Relations |
| BioSample |
SAMN11569835 |
| SRA |
SRX5785810 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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