Scenedesmus bijugus
Mate Pair sequencing library was prepared with Illumina-compatible Nextera Mate Pair Sample Preparation Kit (Illumina Inc., Austin, TX, U.S.A.) at Genotypic Technology Pvt. Ltd., Bangalore, India. About 4 ug of genomic DNA was simultaneously fragmented and tagged with mate pair adapters in a transposon based tagmentation step. Tagmented DNA was then purified using AMPure XP magnetic beads (Beckman Coulter, U.S.A.) followed by strand displacement to fill gaps in the tagmented DNA. Strand displaced DNA was further purified with AMPure XP beads before size-selecting 8-11 kb fragments on Low melting agarose gel. Size selected fragments were circularized in an overnight blunt-end intramolecular ligation step which will result in circular DNA with the insert flanked mate pair adapter junction. Circularized DNA was sheared using Covaris S220 sonicator (Covaris, Woburn, Massachusetts, USA) to generate fragment size distribution from 300 bp to 1000 bp. Sheared DNA was purified to collect the Mate pair junction positive fragments using Dynabeads M-280 Streptavidin magnetic beads (Thermo Fisher Scientific, Waltham, MA, U.S.A.). Purified fragments were end-repaired, adenylated and ligated to Illumina multiplex barcode adaptors as per Nextera Mate Pair Sample Preparation Kit protocol. Adapter-ligated DNA was then amplified for 15 cycles of PCR using Illumina-compatible primers. Final PCR product (sequencing library) was purified with AMPure XP beads, followed by library quality control check. Illumina-compatible sequencing library was quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent TapeStation.
Green_Algae_Ext1_3-5