title: GSM5510378: Bigelowiella_natans_Hi-C; Bigelowiella natans; Hi-C
library_construction_protocol: B. natans cells were first crosslinked using 37% formaldehyde (Sigma) at a final concentration of 1% for 15 minutes at room temperature. Formaldehyde was then quenched using 2.5 M Glycine at a final concentration of 0.25 M. Cells were subsequently centrifuged at 2,000 g for 5 minutes, washed once in 1x PBS, and stored at -80C.  Cell lysis was initiated by incubation with 250 uL of cold Hi-C Lysis Buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) on ice for 15 minutes, followed by centrifugation at 2,500 g for 5 minutes, a wash with 500 uL of cold Hi-C Lysis Buffer, and centrifugation at 2,500 g for 5 minutes. The pellet was the resuspended in 50 uL of 0.5% SDS and incubated at 62C for 10 minutes. SDS was quenched by adding 145 uL of H$_2$O and 25 uL of 10% Triton X-100 and incubating at 37C for 15 minutes. Restriction digestion was carried out by adding 25 uL of 10x NEBuffer 2 and 100 U of the MluCI restriction enzyme (NEB, R0538) and incubating for >=2 hours at 37C in a Thermomixer at 900 rpm. The MluCI restriction enzyme was chosen as more suitable for the highly AT-rich nucleomorph genome. The reaction was then incubated at 62C for 20 minutes in order to inactivate the restriction enzyme. Fragment ends were filled in by adding 37.5 uL of 0.4 mM biotin-14-dATP (ThermoFisher Scientific, # 19524-016), 1.5 uL each of 10 mM dCTP, dGTP and dTTP, and 8 uL of 5U/uL DNA Polymerase I Large (Klenow) Fragment (NEB M0210). The reaction was the incubated at 37C in a Thermomixer at 900 rpm for 45 minutes. Fragment end ligation was carried out by adding 663 uL H$_2$O, 120 uL 10x NEB T4 DNA ligase buffer (NEB B0202), 100 uL of 10% Triton X-100, 12 uL of 10 mg/mL Bovine Serum Albumin (100x BSA, NEB), 5 uL of 400 U/uL T4 DNA Ligase (NEB M0202), and incubating at room temperature for $\geq$4 hours with rotation.  Nuclei were then pelleted by centrifugation at 2,000 g for 5 minutes; the pellet was resuspended in 200 uL ChIP Elution Buffer (1% SDS, 0.1 M NaHCO3), Proteinase K was added, and incubated at 65C overnight to reverse crosslinks.  After addition of 600 uL 1xTE buffer, DNA was sheared using a Covaris E220 instrument. DNA was then purified using the MinElute PCR Purificaiton Kit (Qiagen #28006), with elution in a total volume of 300 uL 1x EB buffer. For streptavidin pulldown of biotin-labeled DNA, 150 uL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) were separated on a magnetic stand, then washed with 400 uL of 1x TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20). The beads were resuspended in 300 uL of 2x Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), the sonicated DNA was added, and the beads were incubated for >=15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 600 uL of 1x TWB, and heated at 55C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55C incubation were repeated.  Final libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645) as follows. End repair was carried out by resuspending beads in 50 uL 1x EB buffer, and adding 3 uL NEB Ultra End Repair Enzyme and 7 uL NEB Ultra End Repair Enzyme, followed by incubation at 20C for 30 minutes and then at 65C for 30 minutes.  Adapters were ligated to DNA fragments by adding 30 uL Blunt Ligation mix, 1 uL Ligation Enhancer and 2.5 uL NEB Adapter, incubating at 20C for 20 minutes, adding 3 uL USER enzyme, and incubating at 37C for 15 minutes.  Beads were then separated on a magnetic stand, and washed with 600 uL TWB for 2 minutes at 55C, 1000 rpm in a Thermomixer. After separation on a magnetic stand, beads were washed in 100 uL 0.1 x TE buffer, then resuspended in 16 uL 0.1 x TE buffer, and heated at 98C for 10 minutes.  For PCR, 5 uL of each of the i5 and i7 NEB Next sequencing adapters were added together with 25 uL 2x NEB Ultra PCR Mater Mix. PCR was carried out with a 98C incubation for 30 seconds and 12 cycles of 98C for 10 seconds, 65C for 30 seconds, and 72C for 1 minute, followed by incubation at 72C for 5 minutes.  Beads were separated on a magnetic stand, and the supernatant was cleaned up using 1.8x AMPure XP beads.  Libraries were sequenced in a paired-end format on a Illumina NextSeq instrument using NextSeq 500/550 high output kits (either 2x75 or 2x36 cycles).