HT115 (D3) bacteria containing L4440 vector with DNA targeting Renilla luciferase
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
Synchronized L4 animals were washed 5 times in M9 buffer and collected on ice. Nuclei were isolated using a glass Dounce homogenizer with 50 strokes tight-fitting insert in buffer A (15 mM Tris–HCl pH7.5, 2 mM MgCl2, 340 mM sucrose, 0.2 mM spermine, 0.5 mM spermidine, 0.5 mM phenylmethanesulfonate [PMSF], 1mM DTT, 0.1% Triton X-100 and 0.25% NP-40 substitute). The debris were removed by spinning at 100×g for 5 min and nuclei were counted by Methylene blue staining. 100.000 nuclei per sample were pelleted by spinning at 1000×g for 10 min and proceeded immediately to transposition step of the ATACseq protocol. Library preparation was done following a protocol published in Buenrostro et al. 2013. Cell pellet was resuspended in the transposase reaction mix (25 μL 2× TD buffer, 2.5 μL transposase) (Illumina Nextera DNA Library preparation kit, #FC-121-1030). The transposition reaction was carried out for 60 min at 37ºC. The samples were purified using Zymo DNA Clean & Concentrator kit; following purification, library fragments were amplified using NEBNext PCR master mix and custom Nextera PCR primers. Libraries were amplified for a total of 12 to 14 cycles and sequenced using paired-end-sequencing length of 75 nucleotides using NextSeq 500/550 High Output v2 kit (Illumina) on a NextSeq500 machine (Illumina) following manufacturer's protocol.