GSM8988492: Kc_RNAPIIS5p_(240820_BT_Dm_3417); Drosophila melanogaster; OTHER
CUT&Tag chromatin profiling To perform CUT&Tag [Kaya-Okur et al 2019] with Drosophila tissue, we dissected 20 imaginal wing discs from male 3rd instar larvae in PBS buffer, and transferred them to a tube containing Accutase (Stemcell Technologies, #07920) at 25 ̊C for 30 minutes. We then added an equal volume of 30% BSA to block proteases, and ran the material through a 30 1⁄2 gauge needle once to dissociate tissue. Tissue suspensions were divided between 4-8 reaction tubes, and bound with BioMag Plus ConA (Bangs Inc., #531) magnetic beads. For CUT&Tag with Drosophila Kc167 cells and human K562 cells, 50,000 cells were bound to ConA beads for each sample, and then lightly fixed with 0.1% formaldehyde at room temperature for 1 minute. Tissue and cell culture samples were then incubated with the following CUT&Tag solutions sequentially: primary antibodies diluted in Wash+ buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% triton-X100, 2 mM EDTA, 1% BSA, with Roche cOmplete protease inhibitor) overnight at 4 ̊C, secondary antibodies (in Wash+ buffer) for 1 hour at room temperature, and then incubated with pAGTn5 (Epicypher 15-1017) in 300Wash+ buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.05% triton-X100 with cOmplete protease inhibitor) for 1 hour. After one wash with 300Wash+ buffer, tissue samples were incubated in 300Wash+ buffer supplemented with 10 mM MgCl2 for 1 hour at 25 ̊C to tagment chromatin. Cell culture samples were incubated with CUTAC buffer (10 mM TAPS pH 8.5, 20% dimethylformamide) supplemented with 5 mM MgCl2 for 1 hour at 25˚C for tagmentation. Samples were washed with 10 mM TAPS pH 8.5 and DNA was released with 0.1% SDS, 0.012 U/μL thermolabile protease K (NEB P8111S) in 10 mM TAPS at 37 ̊C and inactivated at 55 ̊C. Libraries were enriched with 14 cycles of PCR and sequenced in dual-indexed paired-end mode (PE50) on the Illumina NextSeq 2000 platform at the Fred Hutchinson Cancer Center Genomics Shared Resource. Paired-end reads were mapped to Drosophila (dm6) or human (hg19 February 2019) assemblies using Bowtie2 using parameters, e.g.: --end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700. PMID 31036827