title: GSM5830476: Rad21KO primary dissociated cortical neuron treated with TTX/D-AP5 Rep2 5C; Mus musculus; OTHER
library_construction_protocol: Neuronal cultures were fixed in 1% formaldehyde for 10 minutes (room temp) via the addition (1:10 vol/vol) of the following fixation solution: 50 mM Hepes-KOH (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 11% Formaldehyde. Fixation was quenched via the addition of 2.5 M glycine (1:20 vol/vol) and scraped into pellets. Each pellet was washed once with cold PBS, flash frozen, and stored at -80oC. For each condition, in situ 3C was performed on 2 replicates of 4-5 million cells as described (Beagan et al., 2020). Briefly, cells were thawed on ice and resuspended (gently) in 250 L of lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) with 50 L protease inhibitors (Sigma P8340). Cell suspension was incubated on ice for 15 minutes and pelleted. Pelleted nuclei were washed once in lysis buffer (resuspension and spin), then resuspended and incubated in 50 L of 0.5% SDS at 62oC for 10 min. SDS was inactivated via the addition of 145L H2O, 25 uL 10% Triton X-100, and incubation at 37oC for 15 min. Subsequently, chromatin was digested overnight at 37oC with the addition of 25 L 10X NEBuffer2 and 100U (5 L) of HindIII (NEB, R0104S), followed by 20 min incubation at 62oC to inactivate the HindIII. Chromatin was re-ligated via the addition of 100 L 10% Triton X-100, 120 L NEB T4 DNA Ligation buffer (NEB B0202S), 12L 10 mg/mL BSA, 718 L H2O, and 2000 U (5 L) of T4 DNA Ligase (NEB M0202S) and incubation at 16oC for 2 hours. Following ligation nuclei were pelleted, resuspended in 300 L of 10 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 1% SDS, plus 25 L of 20 mg/mL proteinase K (NEB P8107), and incubated at 65oC for 4 hours at which point an additional 25 L of proteinase K was added and incubated overnight. 3C templates were isolated next day via RNaseA treatment, phenol-chloroform extraction, ethanol precipitation, and Amicon filtration (Millipore MFC5030BKS). Template size distribution and quantity were assessed with a 0.8% agarose gel. 5C primers were designed according to the double-alternating design scheme using the My5C primer design software (http://my5c.umassmed.edu/my5Cprimers/5C.php)10 with universal “Emulsion” primer tails. Regions were designed to capture TAD structures immediately surrounding the genes of interest (Bdnf, Fos, Arc, Neurexin-1, Neuroligin-3, Synaptotagmin-1) in published mouse cortex HiC data. 5C reactions were carried out as previously described. 600 ng (~200,000 genome copies) of 3C template for each replicate was mixed with 1 fmole of each 5C primer and 0.9 ug of salmon sperm DNA in 1x NEB4 buffer, denatured at 95oC for 5 min, then incubated at 55oc for 16 hours. Primers which had then annealed in adjacent positions were ligated through the addition of 10 U (20 uL) Taq ligase (NEB M0208L) and incubation at 55oC for 1 hour then 75oC for 10 min. Successfully ligated primer-primer pairs were amplified using primers designed to the universal tails (FOR = CCTCTC TATGGGCAGTCGGTGAT, REV = CTGCCCCGGGTTCCTCATTCTCT) across 30 PCR cycles using Phusion High-Fidelity Polymerase. Presence of a single PCR product at 100 bp was confirmed via agarose gel, then residual DNA <100 bp was removed through AmpureXP bead cleanup at a ratio of 2:1 beads:DNA (vol/vol). 100 ng of the resulting 5C product was prepared for sequencing on the Illumina NextSeq 500 using the NEBNext Ultra DNA Library Prep Kit (NEB E7370) following the manufacturer's instructions with the following parameter selections: during size selection, 70 uL of AMPure beads was added at the first step and 25 at the second step; linkered fragments were amplified using 8 PCR cycles. A single band at 220 bp in each final library was confirmed using an Agilent DNA 1000 Bioanalyzer chip, and library concentration was determined using the KAPA Illumina Library Quantification Kit (#KK4835). Finally, libraries were evenly pooled and sequenced on the Illumina NextSeq 500 using 37 bp paired-end reads.