GSM7061119: Tet-GFP, LBF (third instar larva brain fraction); Drosophila melanogaster; ChIP-Seq
The larval brain fraction (LBF), 300 frozen larval LBFs were thawed on ice and 1 ml of NU-1 buffer (5 mM HEPES-KOH pH 7.9, 5 mM MgCl2, 0.1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 350 mM sucrose, 1mM DTT). 1% formaldehyde was added to NU-1 buffer before use. Samples were homogenized immediately at room temperature using Dounce with a loose pestle 30 times without foaming for 15 minutes. Samples were filtered first through BD Falcon Cell Strainer 70 μm (Cat No.352350) followed by 50 μm Filcon (Cat No. 340603). Samples were quenched with freshly prepared 125 mM glycine incubated for 5 minutes at room temperature on a shaker and transferred to ice for 5 minutes. Samples were centrifuged at 4000 g at 4˚C for 5 minutes. The pellet was washed twice with 1 ml cold PBS and resuspended in 350 µl chilled sonication buffer (50mM HEPES-KOH pH 7.9, 140 mM NaCl, 1mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 1% SDS) and incubated for 20 minutes at 4˚C. Lysates were sonicated as described above and chromatin was stored at - 80˚C until immunoprecipitation. ACCEL-NGS® 1S plus DNA library kit was used to prepare indexed libraries from IP and input DNA. Libraries were pooled respecting equimolarity. Sequencing was performed on Illumina MISeq sequencer in 150 bp paired-end reads.