V3 region sequencing of RE of H salicifolia
For identification of root endobacteria, roots from 4 Naturally grown Hippophae salicifolia were collected in sterile poly bags and stored in refrigerator. The root samples were cut in to thin sections using a sterile surgical blade and grinded in a sterile mortar and pestle to powder. Equal quantity of powdered root sample from each of the four collected samples were mixed and total community DNA was extracted using PowerPlant Pro DNA Isolation Kit (MO BIO Laboratories, Inc.). V3 region was amplified by PCR (950C for 3min, followed by 20 cycles at 950C for 30 sec, 600C for 30 sec, 720C for 30 sec, and ended with a final extension step at 720C for 3 min) using 20 ng extracted metagenomic DNA and V3 region specific primers (341F, 59-CCTACGGGAGGCAGCAG-39 and 518R, 59-ATTACCGCGGCTGCTGG-39) liagated with illumina adapter sequences 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' respectively. The amplified product was then gel purified. In the next step, using purified amplicon above, index PCR  (950C for 3min, followed by 8 cycles at 950C for 30 sec, 550C for 30 sec, 720C for 30 sec, and ended with a final extension step at 720C for 5 min) was performed to attach dual indices and Illumina sequencing adapters using the Nextera XT Index Kit. In the next step AMPure XP beads were used to clean up the final library before quantification using fluorometric quantification method. The library was then denatured using MiSeq v3 reagent kits. 20pM of denatured library were mixed with 20pM of denatured PhiX Control libaray prepared in the same way. The denatured library were then loaded in MiSeq v3 reagent cartridge and used for cluster generation and sequencing. After sequencing, the raw reads are demultiplexed, adapter sequences are removed, quality checked to eliminate low quality sequences. The cleaned high quality raw reads are then used for downstream analysis.
SUB4336922
C_V3