Bluepippin dna size selection system

In this study, we used immortalized B cells derived from Japanese subjects that were distributed by the Japanese Collection of Research Bioresources (Japanese B cell DNA bank), the National Institute of Biomedical Innovation, Health and Nutrition. To collect more information about the Y chromosome, 258 of the 270 control samples are male. For SMRTbell library preparation, B cell DNA was sheared twice using a Diagenode’s Megaruptor 2 (Diagenode, Denville, NJ, USA) set to 25 kb, and purified using a 1× volume ratio of AMPure PB beads (Pacific Biosciences, Menlo Park, CA, USA). DNA sizing was checked on the FEMTO Pulse (Agilent) using the Genomic DNA 165 kb kit on extended mode. SMRTbell libraries for sequencing were prepared using the Procedure & Checklist—Preparing HiFi SMRTbell Libraries using the SMRTbell Express Template Prep Kit 2.0 protocol. Briefly, the steps included DNA repair, overhang adapter ligation using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences), 10-kb cutoff size selection using the BluePippin DNA Size Selection System by Sage Science, and binding to polymerase using the Sequel II Binding Kit 2.2 (Pacific Biosciences). Sequel II CCS/HiFi libraries were sequenced using the Sequel II Sequencing Plate 2.0 and SMRT Cells 8M (Pacific Biosciences) with a movie length of 30 h.

For Illumina sequencing, we extracted genomic DNA from bacterial isolates on a JANUS automated workstation (PerkinElmer, https://www.perkinelmer.com) by using Chemagic magnetic bead technology, according to the manufacturer’s instructions. We prepared DNA libraries by using a NexteraXT DNA preparation kit (Illumina, https://www.illumina.com) and performed 2 × 100 bp sequencing on the NextSeq 500 platform (Illumina), as previously described (16 (link)). Four representative C. jejuni isolates also underwent whole-genome sequencing on the Pacific Biosciences, Inc., RS II platform (https://www.pacb.com). For this, genomic DNA was extracted from overnight cultures by using the Genelute bacterial genomic DNA kit (Sigma Aldrich). DNA libraries were prepared according to the 20 kb Template Preparation using the BluePippin DNA Size Selection system protocol (Pacific Biosciences, Inc). Sequence data are available from GenBank BioProject ID PRJNA520992 and PubMLST (https://pubmlst.org/campylobacter) nos. 70207–12, 70229, 70230, 70232, 70233, 70252, 70253, and 78631–845.

Genomic DNAs from the three inbred medaka strains were used to prepare SMRTbell libraries. DNA was sheared, using a g-TUBE device (Covaris Inc., Woburn, MA, USA) operating at 4,300 rpm and purified using a 0.45 × volume ratio of AMpure beads (Pacific Biosciences, Menlo Park, CA, USA). SMRTbell libraries for sequencing were prepared using the “20 kb Template Preparation using BluePippin Size Selection System (15 kb Size Cutoff)” protocol. Briefly, this features (1) DNA repair; (2) blunt ligation with hairpin adapters employing the SMRTbell template Prep Kit 1.0 (Pacific Biosciences); (3) size selection using the BluePippin DNA size selection system of Sage Science; and (4) binding of DNA fragments to Polymerase P6 using the DNA Sequencing Reagent 4.0 (Pacific Biosciences). SMRTbell libraries were sequenced on a SMRT Cell (Pacific Biosciences) using magnetic bead loading and P6-C4/P5-C3/P4-C2 chemistry. Sequence data were collected with the aid of a magnetic bead collection protocol. The insert size was 20 kb; “stage start” was enabled, and 240-min movies were run employing PacBio RS Remote. Primary filtering was performed on the PacBio RS II Blade Center server.