GSM9669049: experiment 1: RpA70-EGFP NC14 ChIP for total H3, replicate 1; Drosophila melanogaster; ChIP-Seq
Following ChIP fixation (15 minutes Formaldehyde + PBS-triton-X-100 + Heptane) embryos were sorted by stage using RpA70-EGFP fluorescence observed under a stereomicroscope. Interphase embryos with NC10 or earlier nuclear density were collected for the NC1-10 sample, and late NC14 embryos including those with cephalic furrows, but not ventral furrows, were collected for the NC14 sample. ChIP was performed by initially sonicating chromatin and incubating overnight with a primary antibody. Antibody-DNA complexes were pulled down using protein G dynabeads. Before washing, antibody-DNA complexes were subjected to tagmentation with Illumina Tn5 transposase for 40 minutes at 37°C. Transposed samples were then washed in low and high salt solutions (2x 5' each) followed by one wash in TE buffer. Crosslinks were reversed for 4 hours at 65°C with moderate shaking in the presence of 5 µg/ml ProteinaseK. DNA was cleaned up using a Zymo ChIP Clean and Concentrator kit, and PCR was performed using sets of unique-dual-indexed Nextera PCR primers. Libraries were amplified within the linear range of PCR prior to Ampure clean up. Libraries were pooled as necessary and sent to Admera Health for sequencing on a NovaSeq X platform (paired-end 150 bp reads). Admera returned barcode-split, compressed fastq files for reads 1 and 2.