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| Status |
Public on Sep 13, 2010 |
| Title |
Rn_enrich_21011 |
| Sample type |
SRA |
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| Source name |
tail
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| Organism |
Rattus norvegicus |
| Characteristics |
genotype: F1 animals from mutant vs wildtype
phenotype: unknown
enrichment: yes
tissue: tail
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
One microgram of each individual genomic DNA sample was fragmented to ~ 100 nt double-stranded DNA fragments by sonication in 100 µl of 10 mM Tris buffer pH 8 using Covaris S2 sonicator (6 x 16 mm AFA fiber Tube, duty cycle: 20%, intensity: 5, cycles/burst: 200, frequency sweeping, 6 minutes). After fragmentation, fragments were blunt-ended and phosophorylated at 5'-prime end using End-it Kit (Epicentre) according to the manufacturer’s instructions. Double stranded adapters compatible with SOLiD sequencing (adapter 1: pre-annealed duplex of 5'-CTA TGG GCA GTC GGT GAT-3' and 5'-ATC ACC GAC TGC CCA TAG TTT-3' and adapter 2 (sequence depends on multiplexing); adapter 2 for non- multiplex libraries: pre-annealed duplex of 5'- CCG GGT TCC TCA TTC TCT-3' and 5'- AGA GAA TGA GGA ACC CGG TTT-3', adapter 2 for multiplexed libraries: pre-annealed duplex of 5'-CGC CTT GGC CGT ACA GCA G-3' and 5'-GCT GTA CGG CCA AGG CG-3'; all oligo’s were acquired through Integrated DNA Technologies (Coralville, IA) and pre-annealing was done by mixing complementary oligonucleotides at 500 µM concentration and running on thermocycler with the following program: 95˚C for 3 min, 80˚C for 3 min, 70˚C for 3 min, 60˚C for 3 min, 50˚C for 3 min, 40˚C for 3 min and 4˚C hold), were ligated to the fragmented DNA (~ 1 µg) using Quick ligation kit (New England Biolabs) with 750 mM adaptor 1 and adaptor 2, 150 µl of 2x Quick ligation buffer, 5 µl Quick Ligase in a total volume of 300 µl. Samples were purified using Ampure beads (Agencourt) and amplified in 400 µl of Platinum PCR Supermix with 750 mM of both amplification PCR primers (P1_short: 5'-CTA TGG GCA GTC GGT GAT-3' with P2_short: 5’-CCG GGT TCC TCA TTC TCT -3’ for non-barcoded samples, with Bar_primer: 5’-GCT GTA CGG CCA AGG CG-3’ for multiplexed samples and with BC-primer: 5’-CTG CCC CGG GTT CCT CAT TCT CTN NNN NNN NNN CTG CTG TAC GGC CAA GGC G-3’ for barcoded samples multiplexed before enrichment, where N represent unique barcode sequence for each library), 2.5 U of Pfu DNA polymerase (Stratagene) and 5 U Taq DNA polymerase (Bioline). Before ligation-mediated amplification, the PCR sample was incubated at 72˚C for 5 minutes in PCR mix to perform nick translation on non-ligated 3'-ends. After 6 to 8 cycles of amplification, the library DNA was purified on Ampure beads and the quality was checked on a gel for the proper size range and the absence of adapter dimers and heterodimers. Finally, single samples or equimolar pools of multiplexed barcoded samples were size selected on 4% agarose gel for 125-175bp fraction.
microarray hybridization: DNA was mixed with 31.7 μl of Nimblegen aCGH hybridization solution and denatured at 95˚C for 5 minutes. After denaturing the sample was hybridized for 72 hours at 42˚C on MAUI hybridization station. After hybridization, the array was washed using Nimblegen Wash Buffer Kit according the user’s guide for aCGH hybridization. Elution was performed using 800 μl of elution buffer (10 mM Tris pH 8.0) in an Agilent Microarray Hybridization Chamber at 95˚C for 30 minutes. After 30 minutes the chamber was quickly dissembled and elution buffer was collected. Eluted library DNA was concentrated by speedvac to a volume of 50 μl
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
AB SOLiD System 3.0 |
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| Data processing |
SOLiD sequence tags were mapped to the full genome (Arabidopsis tair8 and rgsc3.4) with the MAQ package 40 (V0.7.1, options: -c, n3, e180, C10)
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| Submission date |
Jun 18, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Michal Mokry |
| E-mail(s) |
m.mokry@umcutrecht.nl
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| Organization name |
Wilhelmina Children's Hospital, University Medical Center Utrecht
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| Street address |
Lundlaan 6
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| City |
Utrecht |
| ZIP/Postal code |
3584 EA |
| Country |
Netherlands |
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| Platform ID |
GPL10462 |
| Series (1) |
| GSE22024 |
Next Gen(etics): targeted genome enrichment and next-generation sequencing enhances phenotype-driven forward genetics and gene-driven reverse genetics |
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| Relations |
| SRA |
SRX025151 |
| BioSample |
SAMN00027332 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM557667_Rn_enrich_21011.bedgraph.gz |
61.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM557667_Rn_enrich_21011.sam.gz |
1.5 Gb |
(ftp)(http) |
SAM |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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