Shot-gun metagenome of Halisarca dujardinii sponge, 2019 (Illumina)
0.5 g of fresh H. dujardinii tissue was transferred to sterile prechilled 35 mm petri dish on ice. The tissue fragment was minced in 1 ml of ice-cold Lysis Buffer (10 mM HEPES pH 7.2, 5 mM MgCl2, 10 mM NaCl, 0.005% NP40) using two disposable scalpels. Tissue was triturated by 10x pipetting with 1 ml tip and transfered to MACS C-tube. Additionally, the petri dish was rinsed with 1 ml Lysis Buffer and it was transferred to C-tube. The tissue was homogenized using gentleMACS Program h_mito_01 in the cold room. The homogenate was incubated for 10 min on ice, and homogenized again using the same program. An equal volume (2 ml) Isotonic Adjustment Buffer (10 mM HEPES pH 7.2, 5 mM MgCl2, 10 mM NaCl, 500 mM Sucrose) was added to MACS C-tube and mixed with homogenate. The homogenate was filtered through a pre-wetted (with 200 uL Isotonic Wash Buffer - 10 mM HEPES pH 7.2, 5 mM MgCl2, 10 mM NaCl, 250 mM Sucrose, DPBS + 1% BSA) 70 m filter into a 15 ml tube. 2 ml Isotonic Wash Buffer was added to MACS C-tube, rinsed around the tube and then rinsed through the filter. The filtered homogenate was centrifuged at 1000xg for 5 min at 4 C. The supernatant was collected (the bacterial fraction) and the pellet (nuclei fraction) was discarded. Bacteria were pelleted by centrifugating the bacterial fraction at 5000xg for 5 min at 4 C. DNA extraction was performed from the bacterial pellet using DNeasy PowerLyzer Microbial Kit (Qiagen, Hilden, Germany) according to the manufacturers instructions, including a bead-beating stage using a FastPrep-24 5G grinder (MP Bio, Santa Ana, CA, USA). A shotgun WGS library preparation was performed at Laboratory "Genomed" Ltd. (Moscow, Russia) using the MGIEasy Fast PCR-FREE FS Library Prep Set (MGI, China) according to the manufacturers protocol. Sequencing was performed using the DNBSEQ-G400 system (MGI, China) in a 150+150 nt paired-end mode.
SUB15321912
Hal_pro