Genomic dna 165 kb kit

A total of 990 blood extracted genomic DNA were analyzed for integrity by calculating their DNA Integrity Number (DIN, threshold set at 10 kb) using Genomic DNA ScreenTape Analysis on a 4200 TapeStation System (Agilent) according to the manufacturer’s instructions. Some high molecular weight genomic DNA samples were further analyzed by highly sensitive high-resolution capillary electrophoresis using the Genomic DNA 165-kb Kit on a Femto Pulse System (Agilent) according to the manufacturer’s instructions.

The quantity and concentration of DNA present was measured in triplicate using a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, Q32853) and a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Q33216). Absorbance values (to measure contamination) were taken using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). Size measurements were taken with either a Tapestation 2200 (Agilent Technologies, Santa Clara, CA, USA) and Genomic DNA Screentape (Agilent Technologies, 5067-5365) and Genomic DNA Reagents (Agilent Technologies, 5067-5366), or a Femto Pulse (Agilent Technologies) and the Genomic DNA 165 kb Kit (Agilent Technologies, FP-1002-0275). To size-evaluate the high molecular weight DNA, GQN (Genomic quality number) analyses of Femto Pulse runs were carried out using Prosize (Agilent Technologies) in gDNA mode; for example, a GQN_50kb of 0.5 indicates that 50% of fragments are longer than 50 kb in the total DNA [19 ].

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.

A single colony of P. fuscovaginae UPB0736 was inoculated in 5 ml of KB broth and incubated overnight at 28°C, 150 rpm. Two ml of bacteria culture were pelleted (10,000 g, 2 minutes). Genomic DNA was isolated with the Wizard^® Genomic DNA Purification Kit (#TM050, Promega corporation, WI, US) following manufacturer instructions. The quality and integrity of the genomic DNA were checked spectrophotometrically (DS-11, DeNovix) and via gel electrophoresis. The DNA was quantified with the QuantiFluor^® dsDNA system (Promega corporation, WI, US) following the manufacturer’s instructions and measured in a plate reader (Infinite 200 Pro M Plex; Tecan, Switzerland). Genomic DNA fragment size was verified via pulsed-field capillary electrophoresis (Femto Pulse System, Agilent, CA, US) with a Genomic DNA 165 kb Kit (Agilent, CA, US). The genomic DNA was sequenced by Eurofins with PacBio (INVIEW De Novo Genome 2.0: 5.1 – 10Mb). Assembly and polishing were performed by the company. The single contig genome of P. fuscovaginae UPB0736 has been deposited in the NCBI database with accession number CP100603.