title: Bacterial community in Bukpuk cave (CBPV3)
design: Bacterial DNA was isolated from the cave sediment samples using the Fast DNA spin kit (MP Biomedical, Solon, OH, USA) and the V3 hypervariable region of the 16S rRNA gene was amplified using 10 pmol/µl of each forward 341F (5’-3’) and reverse 518R (5’-3’) primer. PCR Master Mix will contain 2 μL each primers, 0.5 μL of 40mM dNTP (NEB, USA), 5 μL of 5X Phusion HF reaction buffer (NEB, USA), 0.2 μL of 2U/μL F-540Special Phusion HS DNA Polymerase (NEB, USA), 5 ng input DNA and water to make up the total volume to 25 μL. The PCR conditions was 98°C for 30 sec followed by 30 cycles of 98°C for 10 sec; 72°C for 30 sec and a final extension at 72°C for 5 sec followed by 4°C hold.Paired end Illumina sequencing (2 x 150 bp) was carried out at Scigenome Lab, Cochin, India.