title: GSM8282543: H4K20me1, ChIP, CEW1, early stage synchronized larvae, rep 1; Oscheius tipulae; ChIP-Seq
library_name: GSM8282543
library_construction_protocol: Around 100-150 µL of pelleted larvae was removed from -80 ˚C and washed in FA Buffer (final concentrations of 50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate, 150 mM NaCl, filter sterilized) with 0.1% sarkosyl, 1 mM PMSF, and 1X protease inhibitors (protease inhibitor cocktail set I – Calbiochem, catalog # 539131) added fresh. Larvae were then dounced 30 times in a glass dounce tissue grinder (VWR, catalog number 22877-280) with pestle type B. Sonication was performed in a Biopruptor ® Pico (catalog # B01060010) to obtain fragments between 200 and 800 bp in length (30 seconds on, 30 seconds off, 10-15 cycles). Protein concentration was determined using the Bradford assay (Bio-Rad, catalog # 500-0006). For chromatin immunoprecipitation, 1-2 mg of extract and 3-5 µg of antibody were used (see supplemental file 1), and allowed to rotate overnight at 4 ˚C. nProtein A Sepharose beads (Cytiva, catalog # 17528001) were added and incubation was allowed to continue for 2 hours. Beads were washed with FA buffer, FA buffer – 1 M NaCl, FA buffer – 500 mM NaCl, TEL buffer (final concentrations of 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0, filter sterilized), and TE, respectively. Immunocomplexes were eluted twice with ChIP elution buffer (final concentrations of 1% SDS, 250 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA) at 65 ˚C for 15 min at 1400 rpm. The eluate was then treated with 2 µL of 10 mg/mL proteinase K for 1 hour at 50 ˚C, and reverse crosslinked overnight in a 65 ˚C waterbath. DNA was purified the next morning with Qiagen MinElute PCR Purification Kit (catalog # 28006), and fragment size was checked by agarose gel electrophoresis or TapeStation. The remaining DNA was stored at -20 ˚C. Library preparation was performed as follows. To perform end repair, we used half the ChIP DNA and 10-30 ng of input DNA, T4 DNA Ligase Reaction Buffer (NEB, catalog #  B0202S), 0.4 mM dNTPs, 20 U PNK (Fisher, catalog # FEREK0032), 0.7 U Large Klenow Fragment (NEB, catalog # M0210S), and 6 U T4 DNA Polymerase (Fisher, catalog # FEREP0062) (total volume 33 µL). The reaction was incubated at 20 ˚C for 30 minutes, and was cleaned with Qiagen MinElute PCR Purification Kit (eluted in 21 µL). To perform A-tailing, NEBuffer 2 (catalog # B7002S), 0.2 mM dATP, and 7.5 U of Klenow Fragment (3' → 5' exo-) (NEB, catalog # M0212L) were added to the cleaned DNA (total volume of 25 µL), and incubated for an hour in a 37 ˚C water bath. The reaction was cleaned with Qiagen MinElute PCR Purification Kit (eluted in 17 µL). To perform adapter ligation, Quick Ligation Buffer, Illumina Truseq Adapters (see supplemental file 1), and 2 µL of Quick Ligase (NEB, catalog # M2200L) were added to cleaned DNA (total volume of 40 µL), and incubated for 20 minutes at 23 ˚C. The reaction was size selected and cleaned with Ampure XP beads (Beckman Coulter, catalog # A63881). The eluate was amplified by PCR with Phusion HF Reaction Buffer, 0.5 µM TruSeq forward and reverse primers (see supplemental file 1), 0.2 mM dNTP, and 1 U Phusion High-Fidelity DNA Polymerase (Fisher, catalog F-530L) for a total reaction volume of 50 µL under the following parameters: 98 ˚C for 1 min, 98 ˚C for 30 seconds, 60 ˚C for 30 seconds, 72 ˚C for 30 seconds, repeat steps 2-4 14 times, 72 ˚C for 5 min, 8 ˚C forever. 10 µL of 3 M sodium acetate (pH 5.2) was added to reaction, and the reaction was cleaned with Qiagen MinElute PCR Purification Kit, (eluted in 11 µL). The eluate was gel purified (1.5% agarose gel) to obtain fragments between 250-600 bp using the Qiagen MinElute Gel Extraction Kit (catalog # 28606). Library concentration was determined with the KAPA Library Quantification Kit (catalog # KK4824). Single-end (75 bp) sequencing was performed at the Genomics Core at the Center for Systems and Genome Biology, New York University using the Illumina NextSeq 500 platform.