GSM9535252: Primary spermatocytes, Bearded dragon, HiC, rep2; Pogona vitticeps; Hi-C
In nuclei Hi-C was adapted from previously described protocols.  Briefly, germ cells were incubated in lysis buffer containing 10 mM Tris, 10 mM NaCl, 0.2% Igepal and 1xPIC for 20 minutes at 4ºC. Subsequently, germ cells were incubated with 0.5% SDS in 1xNEBuffer 2 for 10 minutes at 4ºC and the reaction was quenched by adding 10% Triton X-100 and incubating for 30 minutes at 37ºC. Germ cells were then centrifuged at 5,000 xg for 5 minutes at 4ºC and resuspended in 300 µl 1xNEBuffer 2 containing 400 U of MboI. Chromatin was digested overnight at 37ºC and assessed the following day by comparing a digested and a non-digested control. Germ cells were then incubated at 65ºC for 20 minutes to inactivate the restriction enzyme and the reparation mix (0.05mM dCTP, 0.05mM dTTP, 0.5mM dGTP, 0.05mM biotin-dATP, 50U Klenow in 1xNEBuffer 2) was directly added into the cell suspension. Chromatin was repaired and biotinylated for 90 minutes at 37ºC, and then the enzyme was heat-inactivated at 65ºC for 10 minutes. Prior to ligation, germ cells were centrifuged for 5 minutes at 5,000 xg at 4ºC, resuspended in ligation buffer (0.8% Triton X-100, 0.1 mg/ml BSA, 10,000 U T4 DNA ligase in 1xNEB T4 ligase buffer) and incubated overnight at 16ºC. Lastly, germ cells were incubated with RNAse A for 15 minutes at 37ºC, and then with proteinase K overnight at 65ºC to reverse the crosslink, followed by a DNA extraction with Phenol:Chloroform. DNA concentration was quantified by Qubit and quality was assessed by running a 0.8% agarose gel. For library preparation, samples were sonicated into fragments of 300 – 500 bp. The biotinylated fragments were pulled down using Dynabeades MyOne Streptavidin T1 beads, which were pre-washed with 2xbinding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) and incubated with sonicated fragments for 30 minutes in rotation. Beads were then separated on a magnetic rack and washed with 1xbinding Buffer. Ends repair, adaptor ligation and amplification were performed with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® according to manufacturer's recommendations. For size selection, DNA fragments were incubated with AMPure beads in a 1:1 proportion for 10 minutes at room temperature. Beads were then washed twice with 70% ethanol and DNA was eluted with Tris buffer for 10 minutes at room temperature. DNA concentration was quantified by Qubit and fragment size was assessed by running a Bioanalyzer. Libraries were submitted for Illumina sequencing (paired-end 150bp each side on NovaSeq 6000).