GSM6065985: AJ5_14; Mus musculus; OTHER
Briefly, heart and muscle tissues were obtained from wildtype neonate hearts of reciprocal F1 C57BL/6 (B6) and Japanese Fancy [J11] F1 hybrid mice, and J[11] JF1 mice bred to male or female CBR2.3/ mice to generate maternal- and paternal-specific CBR2.3 KO F1 hybrids (CBR2.3/+ mKO and CBR2.3+/ pKO). 3C libraries were generated from nuclei using standard methods and DnpII as the primary restriction enzyme. Before oligonucleotide capture, 3C libraries were fragmented to 200-1000 bp sizes using a second digestion with two spike-ins of NlaIII overnight and in the morning for library prep and sequencing. The xGen Prism DNA library kit with primers (IDT 10006202 & 10005975) and adapters with unique molecular identifier (UMI) sequences were incorporated during adapter ligation to minimize sequence errors and reduce PCR duplication rates (IDT, 10006914). 1 ug of indexed samples were pooled before oligonucleotide capture using biotinylated oligonucleotides designed for the Grb10 and Ddc promoters, CTCF binding sites and putative enhancer sequences using IDT Discovery Pool technology. Probe sequences are provided in Supplementary Table 2. The first hybridization reaction included 5 pools of 4 samples (one of each genotype was included in each pool), with each hybridization pool set up using Mouse-COT DNA and 1.5 pM of Discovery Probes using reagents provided by the xGen Hybridization and Wash Kit according to the manufacturer's instructions (IDT, 1080577). After a 16-hour hybridization step, streptavidin bead pulldown (IDT, 1080577) and multiple bead washes using IDT xGen Hybridization and Wash Kit, PCR amplification of the captured material was performed using KAPA HiFi HotStart Readymix Kit (KAPA Biosystems, KR0370) and library amplification primer (IDT, 1077676) for 14 cycles. A second capture was performed as above, with the exception that it was carried out with one-half of the Mouse-COT DNA and probe concentration used in the first capture. The material was sequenced using the Illumina NOVA-Seq platform with 250-bp paired end reads.