title: GSM9003863: UMI-4C-Seq, Day7 Neurons, ctrl, Mavs Viewpoint, rep1; Mus musculus; OTHER
library_construction_protocol: UMI-4C was performed according to the protocol by Schwartzman et al. with small modifications (Schwartzman et al., 2016). In brief, Day 7 In-vitro differentiated neurons were harvested and collected in 10%FCS/1xPBS and crosslinked for 10 min using 2% formaldehyde. Crosslinked cell pellets were snap-frozen and stored at −80°C until further processing. Frozen pellets were resuspended in 250 µL prediluted DpnII buffer and 7.5 µL preheated 10% SDS and incubated on a thermomixer for 1 h at 37°C, shaking at 900 RPM. After adding 75 µL 10% Triton X-100, the solution was incubated again (1 h, 37°C, 900 RPM). The chromatin was digested using 300 U DpnII (NEB, R0543L) in three stages (100 U for 2 h, 100 U overnight, 100 U for 2 h) at 37°C and 900 RPM. The samples were incubated at 65°C for 20 min to inactivate the restriction enzyme and put on ice. Next, the chromatin was ligated by adding 2000 U of T4 DNA ligase (NEB, M0202M) and 10× T4 DNA ligase buffer to a total volume of 650 µL, and the solution was incubated overnight at 16°C with 300 RPM shaking. Ligated chromatin was de-crosslinked by incubation with 4 µL Proteinase K (20 mg/mL, Qiagen, Cat. 19131) overnight at 65°C and 300 RPM. A 3C template was then purified using 1× Ampure XP beads (Beckman Coulter, A63881). Up to 4 µg 3C template per sample were sheared using microTUBE snap-cap tubes (Covaris, 520045) in a Covaris E220 sonicator to an average fragment length of 300 bp. UMI-4C sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645). For each sample, library prep was performed in up to four parallel reactions with a maximum input of 1000 ng per reaction, including a size selection targeting fragments with a length of 300–400 bp (unless the input was <100 ng) and 4–8 cycles of PCR enrichment depending on the input. Next, a series of nested PCR reactions were performed to enrich for fragments captured in the region of the viewpoints of interest. Both rounds of PCR contained 2 µL 10 mM Illumina enrichment primer 2 (CAAGCAGAAGACGGCATACGA) and either a viewpoint-specific “upstream” (reaction 1, 2 µL 10 mM, see Supp. Table 2) or “downstream” (reaction 2, 2 µL 10 mM, see Supp. Table 2) primer. For each sample, we performed up to eight nested PCR reactions in parallel with an input of 100–200 ng per reaction using the KAPA2G Robust ready mix (Sigma-Aldrich, KK5702) in a volume of 50 µL. PCR program: 3 min at 95°C, 20 cycles (18 cycles for reaction 2) of 15 sec at 95°C, 15 sec at 55°C, 60 sec at 72°C, and final elongation of 5 min at 72°C. Between PCR reactions, the product was cleaned up using 1× AmpureXP beads and eluted in 21 µL. The final PCR product was cleaned up using 0.7× AmpureXP beads and eluted in 25 µL. Reactions per sample were pooled, and library concentration was quantified via qPCR using the KAPA SYBR FAST qPCR master mix (Sigma-Aldrich, KK4602). Final libraries were quality controlled for size and concentration and sequenced on a NovaSeq Illumina sequencer with 100-bp paired-end reads.