Chromium genome library and gel bead kit v 2

Genomic DNA (gDNA) with an adjusted concentration between 1.0 and 1.25 ng/µL was used to prepare the whole-genome sequencing libraries using the Chromium Genome Library and Gel Bead Kit v.2, Chromium Genome Chip Kit v.2, Chromium i7 Multiplex Kit, and Chromium controller according to the manufacturer's instructions (10X Genomics). Genomic DNA was combined with Master Mix, a library of Genome Gel Beads, and partitioning oil to create Gel Bead-in-Emulsions (GEMs) on a Chromium Genome Chip. The GEMs were isothermally amplified with primers containing an Illumina Read 1 sequencing primer, a unique 16-bp 10X barcode, and a 6-bp random primer sequence. Barcoded DNA fragments were recovered for Illumina library construction. The amount and fragment size of post-GEM DNA were quantified prior using a Bioanalyzer 2100 with an Agilent high-sensitivity DNA kit. Prior to Illumina library construction, the GEM amplification product was sheared on an E220 Focused Ultrasonicator (Covaris) to approximately 350 bp. Then, the sheared GEMs were converted to a sequencing library following the 10X standard operating procedure. The library was quantified by quantitative polymerase chain reaction (qPCR) with a Kapa Library Quant kit (Kapa Biosystems–Roche) and sequenced on a NovaSeq6000 sequencer (RRID:SCR_020150) (Illumina) with paired-end 150-bp reads.

High-molecular-weight DNA was extracted from a female (NN05) ovary using the 10x Chromium HMW DNA extraction protocol (10x Genomics). With 1 ng of template, DNA whole-genome sequencing libraries were prepared using the Chromium Genome Library and Gel Bead Kit v.2 (10x Genomics, no. 120258), Chromium Genome Chip Kit v.2 (10x Genomics, no. 120257), Chromium i7 Multiplex Kit (10x Genomics, no. 120262) and Chromium controller (10x Genomics). In total, 761.34 million linked-reads (114 Gb, 2 × 150-bp) were generated providing a raw coverage of ~65×.