title: GSM3578084: IP_Hth_ChIP_wing; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: SELEX-seq: All samples were assembled at RT for 30 mins. Procedures followed as described previously (Slattery, Cell, 2011; Kribelbauer, Cell Rep, 2017). ChIP-seq: Inverted larvae were cross-linked at room temperature (RT) for 10 min in 10 ml 1% formaldehyde solution buffered with 50mM HEPES (pH=8.0). Samples were quenched with 1 ml 2.5M Glycine and washed for 5 minutes in quench-solution (125 mM glycine, in 1X PBS and 0.01% Triton X-100). Inverted and cross-linked larvae were washed twice with Buffer A (10mM HEPES, pH=8.0; 10mM EDTA, pH=8.0, 0.5mM EGTA, pH=8.0; 0.025 % Triton-X) and twice with Buffer B (10mM HEPES, pH=8.0; 200mM NaCl, 1mM EDTA, pH=8.0; 0.5mM EGTA, 0.01 % Triton X-100). Wing discs were detached on ice in Buffer B and transferred into a final volume of 1 ml Buffer C (10mM HEPES, pH=8.0 ;1mM EDTA, pH=8.0; 0.5mM EGTA, pH=8.0). Chromatin was sheared into fragments by using a probe sonicator at 15 % amplitude (total time: 12 min with 15 seconds on and 40 second off intervals) and flash-frozen in liquid nitrogen for storage at -80℃ until further processing. Sheared chromatin was diluted in 5X RIPA dilution buffer (1x RIPA: 140mM NaCl; 10mM HEPES, pH=8.0; 1mM EDTA, pH=8.0; 1 % Glycerol; 1% Triton X-100; 0.1% DOC) and blocked with 10μg of the respective IgG-coated magnetic beads (Dynabeads, ThermoFisher) for 1h at 4℃. Beads were removed with a magnetic stand and supernatant was transferred into a new tube. 10 % of the sample was set aside to serve as an input control. Specific antibody and 1 % of Bovine Serum Albumine (BSA) was added to the remaining chromatin and incubated over night at 4℃. 30 μg of IgG-coated and pre-blocked (with 1 % BSA) Dynabeads were added to each chromatin antibody solution and incubated for another 2 hours. Antibody-bound TF-chromatin complexes were isolated by magnetic separation (5 min on a magnetic stand) and beads were washed twice with 1x RIPA, once with high salt RIPA (500mM NaCl), once with LiCl-Buffer and once with TE (10 mM Tris-Base, pH=8.0; 1mM EDTA, pH=8.0). Beads with chromatin and the input sample were redissolved in 0.5 ml Elution-Buffer (TE with 0.5 % Sodium Dodecyl Sulfate (SDS) and 50mM NaCl) and incubated for 30 min at 37℃ with RNase, followed by 2 hours at 55℃ with proteinase K (ThermoFisher). Remaining DNA-protein complexes were decrosslinked by incubating for 16 hours at 65℃. DNA was separated from the Dynabeads by magnetic separation and purified by phenol:chloroform extraction and DNA precipitation using 1x volume of isopropanol in 100mM ammonium acetate and 1 μl glycogen. Precipitated DNA was redissolved in 30 μl TE. ATAC-seq: Wing imaginal discs of third instar larvae were dissected from a lab stock of yw genotype in Phosphate-Buffered-Saline. Discs were washed in nuclear extraction buffer (NEB, 10nM HEPES pH. 7.5, 2.5mM MgCL2, 10mM KCl) and placed in a 1mL dounce homogenizer (Wheaton) on ice. Discs were treated with 15 strokes of the loose pestle, followed by a 10 minute incubation on ice, then 20 strokes of the tight pestle. Nuclei were counted using a hemocytometer, and 50,000 nuclei were transferred to a fresh Eppendorf containing 1mL of NEB buffer +0.1% tween-20. Following a brief mixing the nuclei were immediately pelleted for 10 min at a speed of 1000xg. The pellet was re-suspended in ATAC transposition buffer as in (Buenrostro et al., 2015) and tagmentation was carried out as previously described (Buenrostro et al., 2015). Amplified libraries were purified, and size-selected using  ampureXP (Beckman) beads for  a double-sided selection. Extrated SELEX-seq libraries were amplified using Illumina compatible amplification primers (Illumina small RNAseq or NEBNext Mulitplex Oligos). ChIP-seq and ATAC-seq libraries were constructed using the NEBNext Ultra DNA Library Prep Kit for Illumina with NEBNext Mulitplex Oligos (one separate index per sample) following standard instructions. Libraries were sequenced either at the NY Genome Center (HiSeq-2000) or at Columbia University (NextSeq 500, 75 bp kit).
attributes.tag: GEO Accession
attributes.value: GSM3578084