GSM7500604: BTC3_OligosCTCF_AA_2; Mus musculus; OTHER
Cells were further lysed by re-suspension in water, and then in 0.5% SDS at 62°C for 10 min Each replicate was split between three tubes, re-suspend in 800 μL 1x DpnII buffer (NEB) with 1.6% TritonX100, and digested with 3 sequential additions of 750 units DpnII enzyme at 37°C 1200rpm over 24 hours. Samples were heat inactivated at 65°C for 20 min, and 3 samples from each replicate combined into 7 ml with 1xT4 DNA Ligase Buffer (NEB), with 1% TritonX100, and 12,000 units of T4 DNA ligase at 16°C overnight. Samples were treated with Protinease K at 65°C overnight and RNase A/T1 (ThermoFisher) at 37°C for 1 hour, before a standard Phenol Chloroform/Chloroform extraction and ethanol precipitation was performed. Complete digestion and ligation was assessed by gel electrophoresis. Purified 3C DNA from each sample was sonicated to 200-400 bp with a Soniprep 150 probe sonicator at 4°C, and purified with a standard Ampure XP Bead protocol (Beckman Coulter) using a 1/1.5 DNA to bead ratio. Two Illumina sequencing libraries were prepared per capture pool replicate, with 6 μg of starting DNA in each, and generated using NebNext DNA Library Prep Kit (NEB), with samples indexed with unique barcodes using NebNext Multiplex Oligos for Illumina (NEB). Two separate capture pools were designed to the following Pax6 locus elements: CTCF/Cohesin binding sites, known and predicted enhancer clusters, and the multiple promoters of three genes in the locus Pax6, Elp4 and Rcn1. Capture oligos were designed to each end of the targeted DpnII fragments as described by in (Davies, 2016), and each synthesised as a separate 4 nM reaction, with a 5'-Biotin label on a 120 bp Ultramer (IDT). All capture oligos from each of the two capture pools were mixed at equimolar amounts and pooled to a final concentration of 13 pmol in a volume of 4.5 μL per sequence capture. Libraries where sized and quality controlled on a D1000 Tapestation tape (Agilent). NG Capture-C sequence capture was performed using SeqCap EZ HE-Oligo Kit A or B (dependent on the multiplex barcode) and SeqCap EZ Accessory Kit (Nimblegen) as described in (Davies, 2016), using each of the two capture pools, with 1.5-2 μg 3C library DNA per hybridisation reaction. Each hybridisation reaction was performed on a thermocycler at 47°C and incubated for between 66 and 72 hours. Each hybridisation reaction was then bound to streptavidin beads from SeqCap EZ Pure Capture Bead Kit and washed with SeqCap EZ Hybridization and Wash Kit (Nimblegen), following the manufacture's protocol. Hybridization reactions were split into two and libraries re-amplified using Post LM-PCR oligos (Nimblegen) and Q5 High-Fidelity DNA polymerase (NEB) directly from the beads, and then the DNA was purified using Ampure XP Bead 1/1.8 DNA to bead ratio. A second hybridisation reaction was performed as above on the re-amplifed 3C libraries with two reactions pooled together (~1 μg in each) and incubated for 22-24 hours. Washed and re-amplified double captured libraries where sized and quality controlled on a D1000 Tapestation tape (Agilent), and paired-end sequenced on an Illumina NovaSeq 6000.