Pacbio sre kit

DNA extractions from 5 million cells of HG002 were prepared using PacBio Nanobind CBB kit (SKU 102-301-900) according to UHMW DNA Extraction Cultured Cells protocol (EXT-CLU-001). Standard pipette tips were used to generate a homogenous sample before DNA shearing. DNA was sheared to a target size of 50kb on Megaruptor^® 3. Samples were normalized to 100 μL volume at 50 ng/μL concentration and sheared at speed 27 using Megaruptor shearing kit (E07010003). DNA size was assessed post shearing on Agilent Femto Pulse System using gDNA 165kb Analysis Kit (FP-1002-0275). Post shearing, DNA size selection was performed using PacBio SRE kit (SKU 102-208-300) following manufacturer’s recommendations. Libraries were prepared using Oxford Nanopore Ligation Sequencing Kit V14 (SQK-LSK114) according to Oxford Nanopore protocol GDE_9161_v114_revK_29Jun2022. The libraries were sequenced on Flowcell R10.4.1 on PromethION for 96 hours. Flowcells were washed using the Flowcell Wash Kit (EXP-WSH004) every 24 hours. Fresh libraries were loaded after each wash.

High molecular weight (HMW) was extracted using PacBio’s Nanobind CBB Kit (Pacbio, 102-301-900) from 2 × 10⁶ cells with the Nanobind adherent cultured cells protocol. After extraction, DNA concentration was quantified using the Qubit dsDNA BR assay (Invitrogen, Q32850), sized with a Femto Pulse System (Agilent, M5330AA), and size selected with the PacBio SRE Kit (Pacbio, SKU 102-208-300). Following quality control, the extracted DNA was sheared to a target size of 18–20 kb using the Megaruptor 3 (Diagenode, B060100003). After confirmation of correct sizing, the library preparation was performed SMRTbell prep kit 3.0 (PacBio, 102-141-700) with a PEG wash. The library was sequenced on a Revio flow cell with a 24 h movie time.