GSM6128173: JLT2_7; Mus musculus; OTHER
Briefly, muscle tissues were obtained from wildtype adult or neonate reciprocal F1 C57BL/6 (B6) and C7 F1 hybrid mice, and C7 mice bred to male or female mice with ccd knock out (KO) to generate maternal- and paternal-specific ccd KO F1 hybrids (mKO and pKO). Capture-C was performed simultaneously with the Capture-C described by Juan et al. 2022 and followed previously described methods (Davies et al., 2016). The samples described here included nuclei isolated from neonatal and adult hindlimb muscle obtained from reciprocal hybrid wildtype mice (BxC and CxB) and mice with the maternal and paternal DCCDIvr allele (CCDIvrD/+ and CCDIvr+/D, respectively, where '+' is the wildtype C allele). 3C libraries were generated from nuclei using standard methods (Naveh et al., 2021) 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 multiple CTCF binding sites across the Igf2/H19 locus (including the ICR), Igf2 promoters and previously identified and putative enhancer skeletal muscle enhancers using IDT Discovery Pool technology. Probe sequences are provided in Supp. Table 3.1. The first hybridization reaction included five pools describes by Juan et al. and two additional pools described herewith (one pool assayed the four neonatal muscle samples; the other pool assayed the six adult muscle [see Supp. Table 3.2]), 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.