GSM8384538: Midbrain, C1-C4flxflx;CRE_rep1; Mus musculus; OTHER
To stop fixations, Glycine was added at final concentration of 0.13M and incubated for 5 minutes at RT and 15 minutes on ice. Cells were then washed once in cold PBS, centrifuged at 2500g 4oC for 5 mins (these centrifugation conditions were used for all washes following fixation) and pellets frozen at -80oC. Thawed cell pellets were then lysed (10mM Tris-HCL pH8, 50mM NaCl, 5mM MgCl2, 1mMCaCl2, NP-40 and Roche Complete EDTA-free Sigma #11836170001) and digested with Micrococcal nuclease (LS004798, Worthington Biochem). MNase was titrated with 1 million cells at 3U, 5U and 10U for 20 min at 37 °C in 100μl. Then, the micrococcal nuclease reaction was carried out with 5 million cells per replicate, and the micrococcal nuclease step was scaled up to 500 μL with the optimal concentration of micrococcal nuclease determined with titration experiment. Phosphorylation of DNA ends was done for 30 minutes at 37 °C using T4 Polynucleotide Kinase (NEB M0201), 3' exonuclease activity of Klenow polymerase (NEB M0210) was done also at 37 °C for 30 minutes with. Biotin incorporation with dATP and dCTP (Jena Biosciences NU-835-BIO14-L and NU-809-BIOX-L) was done at 25 °C for 90 minutes. T4-mediated proximity ligation was always done overnight with T4 ligase (NEB M0202M). Proteinase K (NEB P8107S) incubation was also performed overnight at 65 °C. After phenol/chloroform/iso-amyl alcohol extraction, the sample was split into two equal aliquots and was purified on two Zymo DNA clean and concentrator kit columns, eluted with 25 μL (preheated to 70 °C) elution buffer then pooled. Samples were loaded onto 3% TBE NuSieve GTG agarose gel in 4 separate wells. After excision of fragments containing dinucleosomes from each lane (>220bp,<400bp), samples were purified using a Zymo Gel DNA Recovery kit to extract DNA. DNA fragments were not polished prior to streptavidin binding. For biotin purification, 50 μL of Streptavidin C1 beads (Thermo 65002) were used per sample. Libraries were prepared using the KAPA Biosystems HyperPrep kit (Roche KK8502) and Illumina primers. After running a small-scale PCR to determine the optimal number of cycles for the required yield, 4 50μL PCR reactions were set up for each sample as instructed by manufacturers. 200 μL was transferred to a new tube and incubated with 0.9x SPRI beads. Samples were sequenced by 50 bp pair- end sequencing with a NovaSeq 6000 with an SP100 kit by the NICHD Molecular Genomics Core. Region capture of Micro-C samples was done as described previously (Goel et al., 2023) and as recommended by the Twist Bioscience's Standard Hybridization Target Enrichment Protocol with a few modifications. Specifically, to increase stringency, all washes steps were done at 70 °C instead of 48 °C and room temperature. The regions covered by probes can be found in TableS1. While biotinylated probes used in cells were of 120bp, the probes used in embryonic material were of 80bp. Library-prep with material on the beads was done using the Hyper Prep kit (Roche KK8502). For promoter Capture Hi-C, 1µg of Hi-C library per sample was hybridized with 120bp biotinylated oligos using the SeqCap EZ kits (Roche). Following washes, material was amplified by PCR using the Kapa polymerase from the Hyper Prep kit (Roche KK8502). Material from different samples was then combined and 1µg of pooled libraries was recaptured. Probes used for these assays can be found in Table S1. Paired end 50