GSM3966749: Bcl11b biallelic trial 2; Mus musculus; OTHER
CUT&RUN experiments were carried out as previously described (Skene et al, 2018) with the following modifications: 1-2.5x10^5 cells were isolated by FACS as described in sections above, bound to Concanavalin A coated magnetic beads (Bangs Laboratories), and  permeabilized with 0.025% (wt/vol) digitonin. Permeabilized cells were incubated overnight at 4C with 5ug of anti-H3K27me3 (Active Motif 39156) and then washed before incubating with protein A-MNase fusion protein (a gift from Steven Henikoff) for 15 minutes at room temperature. After washing, cells were incubated in CaCl2 to induce MNase cleavage activity for 30 minutes at 0oC. The reaction was stopped with 2XSTOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 mg/mL RNase A and 40 mg/mL glycogen) with 2pg of yeast spike-in DNA per sample. Histone-DNA complexes were isolated from insoluble nuclear chromatin by centrifugation and DNA was extracted with a NucleoSpin PCR Clean-up kit (Macherey-Nagel). Library preparation from CUT&RUN products was completed with KAPA Hyper Prep Kit (KAPA Biosystems) following standard protocol with PCR amplification settings adjusted so that annealing and extension steps are combined into one step at 60oC for 10s. Library products were size selected to be within 200 - 300 bp range using AMPure beads (Agencourt). Libraries were sequenced using an Illumina MiSeq system with paired-end 25 bp sequencing read length and TruSeq primer standard for approximately 5 millions reads per sample.