|
| Status |
Public on Sep 20, 2016 |
| Title |
Dam repl 1 |
| Sample type |
SRA |
| |
|
| Source name |
Kc167 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: Kc167
cell type: drosophila melanogaster dorsal closure stage embryonal cell line
|
| Treatment protocol |
20x106 wild-type Kc167 cells were transfected in duplicates by electroporation with 20 µg plasmids expressing Dam-HP1 or Dam (van Steensel & Henikoff, Nat Biotech, 2000; PMID: 10748524) only and collected two days after transfection.
|
| Growth protocol |
Kc167 cells were cultured at 23.5°C in Shields and Sang M3 Insect Medium (Sigma-Aldrich) with 0.25% Bacto Peptone (BD), 0.1% Yeast Extract (BD), 5% heat-inactivated FBS (Thermo Scientific) and 1% Penicillin/Streptomycin (Thermo Scientific).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
gDNA from 5*106 cells was isolated using ISOLATE II Genomic DNA Kit (Bioline).
500ng gDNA were digested with DpnI (10 U, New England Biolabs) in CutSmart buffer in a total volume of 20 µl at 37°C for 8 h. Reaction was terminated by heat-inactivation at 80°C for 20 min. Fragments were ligated to 12.5 pmol DamID adapters using T4 ligase (2.5 U, New England Biolabs) in T4 ligase buffer in a total volume of 25 µl incubated at 16°C for 16 h. The reaction was heat-inactivated for 10 min at 65°C. Products were then digested with DpnII to remove partially methylated fragments. DpnII buffer and DpnII (10 U, New England Biolabs) were added in a total volume of 80 µl and incubated at 37°C for 1h. 20µl of DpnII-digested products were amplified by PCR. PCR products were cleaned up using the ISOLATE II PCR and Gel Kit (Bioline) according to the manufacturer’s instructions and eluted in 26µl H2O. Fragment ends were blunted using the End-It™ DNA End-Repair Kit (Epicentre) in a 50µl reaction according to the manufacturer’s instructions. Products were cleaned up using the ISOLATE II PCR and Gel Kit (Bioline) and eluted in 26µl H2O. For adding a 3’ overhang, fragments were treated with Klenow fragment (3’-5’ exo-, 25 U, New England Biolabs) in buffer 2 with 200µM dATP (Thermo Fisher Scientific) in a 50 µl reaction incubated at 37°C for 30 min. Reaction was terminated by heat-inactivation at 75°C for 20 min. Products were purified using CleanPCR magnetic beads (CleanNA) according to the manufacturer’s instructions and eluted in 20 µl H2O. 260 ng fragments were ligated to 2.5 µM Y-adaptors using T4 Ligase (2.5 U, New England Biolabs) in T4 buffer in a total volume of 10 µl incubated at 16°C for 16 h. Reaction was terminated by heat-inactivation at 65°C for 10 min. Products were purified using CleanPCR magnetic beads (CleanNA) according to the manufacturer’s instructions and eluted in 20 µl H2O. Adapters and indices for next-generation sequencing and multiplexing were added by PCR.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
wild-type Kc167 cells transfected with Dam replicate 1
|
| Data processing |
Reads were filtered for sequences containing DamID-adapter sequence using cutadapt, aligned to drosophila genome release dm3 using Bowtie 2 and matched with GATC-flanked fragments
Genome_build: dm3
Supplementary_files_format_and_content: space-delimited text files containing GATC fragment ID, chromsome, start, end, methylated fragment count
|
| |
|
| Submission date |
Jun 24, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Bas van Steensel |
| E-mail(s) |
b.v.steensel@nki.nl
|
| Phone |
+ 31 20 512 2040
|
| Fax |
+31 20 669 1383
|
| URL |
http://www.nki.nl/nkidep/vansteensel
|
| Organization name |
Netherlands Cancer Institute
|
| Department |
division of Molecular Biology
|
| Lab |
van Steensel group
|
| Street address |
Plesmanlaan 121
|
| City |
Amsterdam |
| ZIP/Postal code |
1066 CX |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL17275 |
| Series (2) |
|
| Relations |
| BioSample |
SAMN05292478 |
| SRA |
SRX1875107 |
