NextSeq 500 sequencing; Methylation profiling of C.elegans to identify CEH-60/PBX binding sites
DamID sampling and library preparation were carried out essentially as described in (Gómez-Salvidar et al. 2016). Strains carrying either the gfp::dam (LSC1722) or ceh-60::dam (LSC1724) transgene were grown under standard conditions. Standard hypochlorite treatment was used to harvest eggs, which were allowed to hatch overnight in M9 buffer. For growing animals for DamID library preparation, four 100 mm NGM plates seeded with Dam-negative E. coli GM119 were used. Dam-negative bacteria do not show GATC methylation, which occurs naturally in most other E. coli strains and would otherwise contaminate C. elegans DamID libraries with bacterial GATC methylation. Young adult or L2 animals were rinsed off plates and 30 µL aliquots were snap-frozen in liquid nitrogen.  Genomic DNA was extracted and purified using the Qiagen DNeasy Blood and Tissue kit according to the manufacturer's instructions. 200 ng of each genomic DNA sample was digested by incubation with 10 units of DpnI in 10 µL for 6 hours at 37°C, followed by inactivation at 80°C for 20 minutes. DpnI-digested DNA was incubated overnight at 16°C with 2 µL ligation buffer, 1 µL Roche T4 DNA ligase and 0.8 µL of double-stranded adaptors (AdRt: CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA, 50 µM, AdRb: TCCTCGGCCG, 50 µM, mixed separately, heated to 95°C and cooled to room-temperature before addition to ligation mix) in a total volume of 20 µL. Ligase was inactivated by incubation for 10 minutes at 65°C. DpnI-digested adaptor-ligated DNA fragments were purified with AgenCourt AMPure XP® beads and a magnetic particle concentrator, after which the sample was digested with DpnII (NEB) by incubation for 1 hour at 37°C, followed by inactivation at 80°C for 20 minutes and another purification step with AgenCourt AMPure XP® beads. DpnII-digested DNA was then amplified using Taq polymerase and 1.25 µL of 50 µM Adr primer (NNNNGGTCGCGGCCGAGGATC). Amplified adaptor-ligated DNA was analyzed by agarose gel electrophoresis and size-selected for amplicons between 400-1200 bp, the expected size of naturally-occurring GATC-flanked reads, using AgenCourt AMPure XP® beads and a magnetic particle concentrator according to the manufacturer's instructions. Using a 0.3 bead-to-sample ratio and selecting the supernatant, amplicons larger than 1200 bp are discarded. Next, using a 0.8 beads-to-sample ratio and selecting the bead-bound DNA, amplicons smaller than 400 bp are discarded. The DNA pool is separated by size through gel electrophoresis to confirm the presence of DNA only in the expected 400-1200 bp range. Adaptor ligated DNA is enriched by PCR and prepared for sequencing using the NEBNext Singleplex oligos for Illumina® by adding 25 µL of NEBNext Q5 Hot Start HiFi PCR Master Mix to 5 µL Index Primer and 5 µL Universal PCR primer to 15 µL of Adaptor-ligated DNA fragments, followed by 8 cycles of PCR amplification as per the manufacturer's instructions. Finally, the sequencing libraries are purified with AgenCourt AMPure XP® beads and size of the amplicons is confirmed by agarose gel electrophoresis and ExperionTM automated electrophoresis system (Bio-Rad).