S220 focused ultrasonicator

3C samples and controls were quantified using Qubit (Invitrogen), run on a 1% agarose gel and tested by qPCR with KAPA SYBR Fast (Sigma) to determine library quality. qPCR primers are in Supplementary Data 1. Only libraries with a digestion efficiency >70% were used for Capture-C. Libraries were either indexed with NEBNext DNA Library Prep Master Mix for Illumina (New England Biolabs) using 6 µg input 3C DNA as previously described following manusfacture’s instructions or using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). When using the Ultra II kit 3 µg 3C material was sonicated to 200 bp using a Covaris S220 Focused Ultrasonicator, and purified using Ampure XP SPRI beads (Beckman Coulter). DNA was eluted into 53 µL with 1 µL used for D1000 TapeStation analysis (Agilent) and 2 µL used for Qubit quantification (Invitrogen). Fifty microliters of DNA ( ≤2 µg) was then indexed with the following modifications; for the End Prep reaction, the 20 °C incubation was lengthened to 45 min, 5 µL of NEBNext Adaptor was added and incubated for 30 min at 20 °C, the USER Enzyme incubation was extended to 30 min (37 °C), and indexing was performed in two reactions with Herculase II Fusion Polymerase (Agilent) using six cycles of amplification.

Using a Covaris S220 Focused-Ultrasonicator, 2 μg of each DNA sample was sheared to an approximate fragment size of 5000 bp and purified using AMPure XP beads (Beckman Coulter). Library preparation was performed using the NEBNext Ultra DNA Library Prep Kit (kit number E7370L, New England Biolabs) and 8 cycles of PCR. Barcode sequences and barcodes assigned to each sample are described in Additional files 31 and 32. Libraries were then quality controlled using a Bioanalyzer high-sensitivity DNA chip and the Agilent 2100 Bioanalyzer system. One technical replicate did not pass quality control before library preparation and was omitted. The samples were then submitted to Novogene (Tianjin, China) for PacBio sequencing which was performed on the PacBio Sequel platform using a 600-min sequencing strategy and three SMRT cells.