GSM8796081: CR_HOXD13_PFL_Shox2Ull_E135_Rep1; Mus musculus; OTHER
After dissociation cells were processed for CUT and RUN following the protocol described by Skene et al. 2018. After a couple of washes, 250K were isolated and used for downstream steps. Cells were incubated with Concavalin A-coted beads (Con A-coated BioMag® Plus beads #86057). Bounded cells were then incubated overnight at 4 °C with the antibody solution containing 0.02% digitonin (Apollo APOBID3301) and 0.5 µl of HOXD13 antibody (Abcam AB229234). Samples were then incubated with the buffer containing the pA-MNase (produced from Addgene pAG/MNase #123461) for 1h on ice. Digestion wash was performed on low-calcium buffer (4 µl of 100 mM CaCl2 in 196 µl of Dig-Wash Buffer) and stopped after 30 min with  2XSTOP Buffer. DNA was precipitated following standard phenol:chloroform:IAA precipitation. Libraries were prepared by the iGE3 Genomic Platform following starting with 5 ng of CUT&RUN-enriched DNA as starting material and processed with the Illumina TruSeq ChIP Library Preparation Kit and the IDT TruSeq RNA UD Indexes v2. No size selection was performed. Library molarity and quality were assessed with the Qubit (Thermofisher Scientific) and Tapestation (Agilent Technologies - DNA High sensitivity chip). Libraries were sequenced on a NovaSeq 6000 Illumina sequencer for PE 50 reads.