title: Illumina gDNA sequencing of Sepia officinalis
design: High molecular weight genomic DNA was isolated from brain tissue (optic lobe) using NEB Monarch gDNA Purification Kit (T3010S). First, 10 mg of flash-frozen tissue were submerged in 100 l of tissue lysis buffer, cut into small pieces and transferred into 1.5 ml tube containing additional 100 l of lysis buffer. Then, 3 l of 20 U/l Proteinase K was added to the tissue sample and incubated for 2 h at 56 C in a thermal mixer with agitation until tissue pieces were completely dissolved. After incubation, tissue lysate was purified from the debris by centrifugation for 3 min at maximal speed (Eppendorf, Germany). Clean supernatant was transferred to a new 1.5 ml tube and incubated with 3 l RNase A for 5 min at 56 C with agitation at full speed. After RNaseA incubation tissue lysate was column purified and eluted with 80 l of elution buffer according to the manufacturer protocol. The purity of gDNA was assessed using A260/280 and A260/230 absorbance ratios measured by NanoDrop spectrophotometer (ThermoFisher Scientific). The integrity of the obtained gDNA was checked using 0,75% agarose gel electrophoresis by loading 100 ng of gDNA sample and using Hind III digested Lambda DNA (NEB#3012) as a marker.DNA sequencing libraries were prepared from 500 ng of high MW gDNA using Illumina DNA PCR-Free Tagmentation Library Prep Kit (20041794) and UD Indexes Set A (20026121). Obtained dual-indexed single-stranded libraries were quantified using the Qubit single-stranded DNA (ssDNA) assay kit (Q10212, ThermoFisher Scientific). 1000 pM of final library were run on P3 reagents and Illumina NextSeq2000 using Illumina DNA PCR-Free Sequencing and Indexing primer (20041797) with 151 bp paired-end reads.