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| Status |
Public on Jul 30, 2018 |
| Title |
Dam_AbdA-1 |
| Sample type |
genomic |
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| Source name |
AbdA DamID, expt 1
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: testis
gender: male
treatment: Dam fused
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| Treatment protocol |
For identifying abd-A and Zfh1 binding regions in the Drosophila testis the fusion protein was expressed from the uninduced minimal Hsp70 promoter of the UAS vector pUAST.
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| Growth protocol |
Dam was fused to the N terminus of abdA and zfh-1 (isoform RB 3165bp) and transgenic flies were generated. As a control for nonspecific Dam activity, transgenic flies expressing the Dam alone were used (Choksi et al., 2006). To express the AbdA-Dam fusion protein, we first generated a pNDam-Myc-abdA construct by cloning abdA in the pNDam-Myc vector (van Steensel et al., 2001) and then subcloned the NDam-Myc-abdA fragment into the pUAST-attB. The large isoform of the abd-A cDNA (abdA-RB) was amplified using the following primers: 5’- CAC CAT GTC AAA ATT TGT TTT CGA TAG -3’ (forward) and 5’- TTA GGA GTT GAC TTT GCT GAC -3’ (reverse). To express the Zfh1-Dam fusion protein, we first generated a pNDam-Myc-zfh1 construct by cloning zfh1 in the pNDam-Myc vector (van Steensel et al., 2001) and then subcloned the NDam-Myc-zfh1 fragment into the pUAST-attB. The large isoform of the zfh-1 cDNA (zfh1-RB) was amplified using the following primers: 5’- CAC CAT GTT GTC CTG TCT GGC GCC -3’ (forward) and 5’- CTA TTC CCT GTA GGG CTT GCA ATA GGA ATA CCG -3’ (reverse). Generation of transgenic flies using the PhiC31-integrase system (Bischof et al., 2013) and balancing of the stock was done by BestGene Inc. (Chino Hills, CA, U.S.A.). Plasmid DNA (pUAST-Dam-Myc-abdA-attB and pUAST-Dam-Myc-zfh1-attB) was injected into embryos of strain attP40 (cytogenetic location of attP-landing site: 25C7) to create an NDam-abdA-RB and an NDam-zfh1-RB fusion. Transgenic flies were balanced over CyO.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Isolation of genomic DNA (gDNA) for DamID: Genomic DNA was extracted from L3 testes, expressing either the NDam-abdA or the NDam-Zfh1 fusion protein and the Dam protein alone using a specific protocol (Tolhuis et al., 2011) and van Steensel personal communication. Testes from NDam-Myc (control), NDam-Myc-abdA transgenic, NDam-Myc-zfh1 and wt flies were dissected in PBS and transferred to a glass well on ice containing ice-cold PBS. Around 200 testes were pooled in 100 μl of lysis (TENS) buffer. 2 μl of Proteinase K (20 μg ml-1) (Roche) were added and incubated for 7 hours at 65°C. Tubes were mixed every 60 min by gentle tapping. RNAs were degraded by RNase A treatment: 1 μl RNase A (100 μg ml-1) (Qiagen) were added and incubated for 30 min at 37°C. An equal volume (200 μl) of phenol: chloroform: isoamyl alcohol (25:24:1, v/v, Invitrogen) was added and mixed well by inversion until a homogenous solution was acquired. Mixture was then applied onto spin phase lock gel light 1.5 ml tube (5PRIME, Inc.) for efficient separation of the two phases. Tubes were prepared by centrifugation for 1 min at 14,000 (Eppendorf Centrifuge-5424). Spin phase lock gel light tubes containing homogenous mixture of aqueous and organic solution were then spun for 5 min at 14,000 rpm. The gel inside the spin phase lock gel light tube then separated the aqueous and organic solution and the upper aqueous solution containing gDNA was transferred to a clean 1.5 ml tube, followed by DNA precipitation. Absence of DNA degradation and of RNA was verified on a 1% agarose gel.
Digestion and PCR amplification of Dam-methylated DNA. DNA digestion and PCR amplification was done essentially as previous described (Papagiannouli et al., 2014). To cut methylated GATC sequences, gDNA (2.5 μg) from each genotype (NDam-Myc, NDam-Myc-abdA, NDam-Myc-zfh1 and wt) was digested with DpnI in a total volume of 10 μl (1x buffer 4, NEB) at 37°C o/n in a PCR machine with a heated lid to avoid evaporation and condensation. For PCR amplification of the DpnI cut DNA sequences, adaptors were ligated to those fragments. Double-stranded adaptors were generated by mixing equal volumes of oligo AdRt (100 μM) (5’-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3’) and oligo AdRb (100 μM) (5’-TCCTCGGCCG-3’)(Vogel et al., 2007) and heating to 100°C for 1 min. Adaptors were then allowed to cool down in about 20 min at RT. 5 μl of DpnI digested DNA (1.25 μg) and 2 μM of adaptors and 5 units of T4 Ligase in a final volume of 20 μl (1x T4 ligase buffer, Fermentas) were incubated for 3 hours at 16°C in a PCR machine. The ligase was inactivated at 65 °C for 10 min. To avoid amplification of fragments between two neighboring methylated GATC, which was not bound by the fusion construct itself, ligated fragments were cut at unmethylated GATC sites using DpnII. 20 μl of ligation product (1.25 μg) were mixed with 5 units of DpnII enzyme in a final volume of 80 μl (1x DpnII buffer, NEB) and incubated for 2 hours at 37°C in a PCR machine with heated lid to avoid evaporation and condensation. The enzyme was not inactivated and digested DNA was directly used for PCR reaction or stored at -20°C. DpnII digested DNA was amplified using following PCR program. The primer (5’-GGTCGCGGCCGAGGATC-3’) was designed to fit to the adaptor(Vogel et al., 2007). Reaction mix contained approximately 313 ng DNA, 0.2 mM dATP, dCTP, dGTP, 0.16 mM dTTP, 0.04 mM dUTP (needed for fragmentation of the DNA when Affymetrix microarrays are used), 1 μM primer and 1.6 μl of Advantage cDNA Polymerase Mix in a total volume of 80 μl (1x Advantage Polymerase Mix Buffer, Clontech). The PCR product was purified using the QIAquick PCR Purification Kit according to manufacturer’s instruction and eluted in 50μl ddH2O. Successful amplification was verified by running a small aliquot on a 1% agarose gel containing a final concentration of 0.5μg/ml ethidium bromide. Targeted DamID (TaDa) in Drosophila 3rd instar testes cyst and germline cells.
Cell-type specific DamID was performed in cyst cells (CySCs and SCCs) and early germline of 3rd instar larval testes for profiling RNA Pol II occupancy in these cells by crossing UAS-LT3-Dam-Pol II and UAS-LT3-Dam control flies to c587-GAL4 (somatic lineage) or Nanos-GAL4 (early germline) drivers. The protocol used for TaDa in L3 testes cyst cells was based on the protocol described above by integrating the modifications suggested by Tony Southall (Southall et al., 2013).
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| Label |
biotin
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| Label protocol |
Affymetrix Processing of DamID DNA to GeneChip Drosophila Tiling Array, was performed as in http://www.flychip.org.uk/protocols/chip/affy_chip.php .
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| Hybridization protocol |
Affymetrix Processing of DamID DNA to GeneChip Drosophila Tiling Array, was performed as in http://www.flychip.org.uk/protocols/chip/affy_chip.php .
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| Scan protocol |
Affymetrix Processing of DamID DNA to GeneChip Drosophila Tiling Array, was performed as in http://www.flychip.org.uk/protocols/chip/affy_chip.php .
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| Description |
processed data file: AbdA.bedGraph
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| Data processing |
CEL files were processed with R function 'BPMAPCelParser' from R package 'rMAT' using bmap file for Dm_tiling2_MR_v01. Probe intensities were quantile normalized with R function 'normalize.Probes' from R package 'Starr’. The ratio between fused Dam and Dam only was calculated with R function 'getRatio' from R package 'Starr’.
BedGraphs were created using R. The columns represent the ratio (4th column) between experiments (fused Dam and Dam only) and genomic position (chromosome = 1st column, start = 2nd column, end = 3rd column).
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| Submission date |
Jul 29, 2018 |
| Last update date |
Jul 30, 2018 |
| Contact name |
Ingrid Lohmann |
| E-mail(s) |
ingrid.lohmann@cos.uni-heidelberg.de
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| Organization name |
Universität Heidelberg, Centre for Organismal Studies
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| Street address |
Im Neuenheimer Feld 230
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| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
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| Platform ID |
GPL6629 |
| Series (1) |
| GSE117833 |
Decoding the regulatory logic of the Drosophila male stem cell system |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM3309959_AbdA_Treatment_1_Dm_tiling2_MR_v01_.CEL.gz |
26.8 Mb |
(ftp)(http) |
CEL |
| Processed data are available on Series record |
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