|
| Status |
Public on Feb 09, 2017 |
| Title |
L1_AluY_Renal2_A498 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
L1 from A498
|
| Organism |
Homo sapiens |
| Characteristics |
mapped te_cell line info: L1_Renal2_A498
cell line: A498
cell line source: Renal
tissue/cell info.: Adenocarcinoma
|
| Growth protocol |
DNA obtained directly from NCI. Cells grown in RPMI 1640 with 5% FBS (Bio Whittaker, not heat inactivated) and 1% L-glutamine. Cells not grown past 80% confluencey for attached cells, or 0,5 x 10-6 cells for suspended. Trypsinize (for attached cells) cells with 5 ml trypsin-EDTA per T162, for 15 min., at 370C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
isolated gDNA for each cell line was obtained from the NCI who used the Qiagen Maxi Kit Spin Protocol to extract DNA according to the manufacturers instructions
|
| Label |
Cy3
|
| Label protocol |
Five micrograms of genomic DNA of each cell line was digested overnight in parallel reactions using four restriction enzymes (AseI, BspHI, HindIII, and Xbal). Sticky ends were ligated to annealed, partially complementary vectorette oligonucleotide adapters. Each template was aliquoted into 3 separate PCR reactions for L1Hs, AluYa5/8, and AluYb8/9 mobile DNA families. Vectorette PCR using the ExTaq Hotstart DNA polymerase and specific forward primers for each mobile element was performed on each of the samples. The PCR amplicons were digested using MseI, MspI and HpyCH4V restriction enzymes to generate smaller fragments and subsequently concentrated. These were then labeled with Cy3-dUTP for L1 and Cy5-dUTP for Alu reactionsusing exo- Klenow polymerase and random 9-mers.
|
| |
|
| Channel 2 |
| Source name |
Alu from A498
|
| Organism |
Homo sapiens |
| Characteristics |
mapped te_cell line info: Alu_Renal2_A498
cell line: A498
cell line source: Renal
tissue/cell info.: Adenocarcinoma
|
| Growth protocol |
DNA obtained directly from NCI. Cells grown in RPMI 1640 with 5% FBS (Bio Whittaker, not heat inactivated) and 1% L-glutamine. Cells not grown past 80% confluencey for attached cells, or 0,5 x 10-6 cells for suspended. Trypsinize (for attached cells) cells with 5 ml trypsin-EDTA per T162, for 15 min., at 370C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
isolated gDNA for each cell line was obtained from the NCI who used the Qiagen Maxi Kit Spin Protocol to extract DNA according to the manufacturers instructions
|
| Label |
Cy5
|
| Label protocol |
Five micrograms of genomic DNA of each cell line was digested overnight in parallel reactions using four restriction enzymes (AseI, BspHI, HindIII, and Xbal). Sticky ends were ligated to annealed, partially complementary vectorette oligonucleotide adapters. Each template was aliquoted into 3 separate PCR reactions for L1Hs, AluYa5/8, and AluYb8/9 mobile DNA families. Vectorette PCR using the ExTaq Hotstart DNA polymerase and specific forward primers for each mobile element was performed on each of the samples. The PCR amplicons were digested using MseI, MspI and HpyCH4V restriction enzymes to generate smaller fragments and subsequently concentrated. These were then labeled with Cy3-dUTP for L1 and Cy5-dUTP for Alu reactionsusing exo- Klenow polymerase and random 9-mers.
|
| |
|
| |
| Hybridization protocol |
The labeled product was hybridized to the Nimblegen HD 2.1 M custom tiled arrays according to the manufacturer’s instructions.
|
| Scan protocol |
Each array was scanned in a microarray core facility using Nimblegen or Agilent instruments. Each scanned array yielded a raw .tff file.
|
| Description |
TIP-chip L1:Alu ratio files were used to detect L1 peaks for L1 and then Alu:L1 ratio files were used to detect Alu peaks
|
| Data processing |
Nimblescan called peaks using a sliding window threshold. A PERL script removed probes overlapping repeats to reduce noise (RepeatMasking). Nimblescan called peaks using a sliding window threshold. Peaks were ranked by the threshold of the log2 transformed ratio of red (Alu) and green (L1) channels or the reciprocal (settings: percent (p) start=90, p step=1, #steps=76, width of sliding window=1500bp, min probes>4, all probes>2)
Peaks were called on a log2 transformed scale of channel1/channel2 for L1 peaks and then channel2/channel1 for Alu peaks. Gff files containing "_532" contain the log2 data for L1 and .gff files containing "_635" contain log2 data for Alu.
|
| |
|
| Submission date |
Jun 27, 2016 |
| Last update date |
Feb 09, 2017 |
| Contact name |
John G Zampella |
| E-mail(s) |
[email protected]
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Dermatology
|
| Lab |
Kathleen Burns
|
| Street address |
733 N Broadway
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21287 |
| Country |
USA |
| |
|
| Platform ID |
GPL21986 |
| Series (1) |
| GSE83756 |
A map of mobile DNA insertions in the NCI-60 human cancer cell panel |
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