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| Status |
Public on Feb 01, 2022 |
| Title |
Dam in spermatocytes, rep 2 |
| Sample type |
SRA |
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| Source name |
Spermatocytes from 3rd instar larvae testes
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Testes
developmental stage: third instar larvae
strain: pUAST-attB-LT3-Dam/bam-Gal4-VP16
biological replicate: 2
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| Growth protocol |
Flies were grown according to standard protocols.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
after in vivo Dam-methylation, genomic DNAs from either ~200 bam∆86 or 3rd instar larvae testes were manually dissected using dissection needles in PBS at 4°C. Isolation of genomic DNA was performed by DNeasy Blood and Tissue kit (Qiagen), following the protocol for cultured cells. DNA was concentrated by centrifugation in 0.5 ml Amicon-Ultra columns (Millipore) for 10 min at 18000 × g and its concentration was measured using a NanoDrop spectrophotometer. For the selective amplification of methylated GATC-GATC fragments, genomic DNA was first digested by DpnI, followed by ligation of adaptors, DpnII digestion and PCR-amplification as described previously (Vogel et al. 2007) with a minor modification: ~0.5 µg instead of 2.5 µg of genomic DNA was used as an input.We applied 16 cycles of PCR (1 min at 94°C, 1 min at 65°C, 2 min at 68°C) for Dam-only and Dam-Lam DNA samples. Adaptors were removed by DpnII digestion for 2 h at 37°C. The amplified methylated DNA fragments were purified using QIAquick PCR Purification kit (Qiagen), concentrated by centrifugation in 0.5 ml Amicon-Ultra columns (Millipore) for 10 min at 18000 × g, and quantified on a NanoDrop spectrophotometer. 0.5 µg of Dam-only and Dam-Lam DNA samples (each in two biological replicates) were subjected to next generation sequencing (NGS) on Illumina HiSeq 2000 resulting in 41-52 million 100 nt single-end reads per sample.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
DamID-seq_Dam_SpCs_rep2
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| Data processing |
Library strategy: DamID-seq
Two biological replicates of Dam-only or Dam-Lam DamID-seq reads were adapter clipped, mapped to the dm3 genomic assembly by bowtie2 (Langmead 2012) and counted by ‘HTSeq-count’ software (Anders et al, 2015) on the 500-bp genomic bins. The resulted counts of Dam-Lam samples were converted to RPM, normalized to the Dam-only samples counts and log2-transformed. The domain calling was performed with the 2-state HMM algorithm as described earlier (Pindyurin et. al, 2018) without taking the heterochromatin portion of dm3 genome build into account.
Genome_build: dm3
Supplementary_files_format_and_content: bedgraph files for Dam and Dam-Lam profiles of individual replicates in spermatogonia (SG) and spermatocytes (SCL) represent read counts per 300-nt bins generated using HTSeq-count software and R via Rstudio.
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| Submission date |
Sep 06, 2020 |
| Last update date |
Feb 01, 2022 |
| Contact name |
Artem Ilin |
| E-mail(s) |
artem.ilin@su.se
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| Organization name |
Stockholm University
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| Department |
MBW
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| Street address |
Svante Arrhenius väg 20C
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| City |
Stockholm |
| ZIP/Postal code |
11418 |
| Country |
Sweden |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE157565 |
Comparison of genome architecture at two stages of male germline cell differentiation in Drosophila |
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| Relations |
| BioSample |
SAMN16070792 |
| SRA |
SRX9084986 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4770610_DAM.SCL.wt.2.bedgraph.gz |
2.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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