title: Single individual sequencing of Hypsibius dujardini genome replicate 4
design: An adult individual was selected and placed in a sterile agar plate with 1% penicillin streptomycin (Invitrogen) prepared with autoclaved Volvic water for 48 h without food to clear the gut content and remove surface microbes. After 48 h, the animal was repeatedly washed with Milli-Q water on a sterile nylon mesh with 30 µm pores (Millipore), and any surface microbes were observed at x500~x1000 magnification under a microscope (VHX-5000, Keyence). The cleaned animal was transferred to a low-binding PCR tube with minimal water carry-over (<2 µl), to which a 200 µl genomic lysis buffer (Quick-gDNA MicroPrep kit, Zymo Research) with 0.5% beta-mercaptoethanol was added immediately. The animal was lysed using three freeze-thaw cycles of -196°C and 37°C incubation, and genomic DNA was extracted following manufacturer’s protocol. Extracted DNA was then sheared to 550 bp target fragments with Covaris M220 using a 15 µl microTube, and the Illumina library was subsequently prepared using a Thruplex DNA-Seq kit (Rubicon Genomics) according to the manufacturer’s instructions. The purified library was quantified using a Qubit Fluorometer (Life Technologies), and the size distribution was checked using TapeStation D1000 ScreenTape (Agilent Technologies). Library with sizes within the broad peak oberseved from TapeStation electropherogram between 400 bp and 1000 bp was selected by manually cutting them out of the agarose and purifying with a NucleoSpin Gel and PCR Clean-up kit (Clontech). The sample was then sequenced using one MiSeq v.3 600 cycles kit (Illumina) as 300 bp paired ends. Adapter sequences were removed using the MiSeq software (Illumina).