title: Pellet_SC175_Rep1
design: We amplified the V3V4 regions of the bacterial 16S rRNA gene using the primers 338F (5-ACTCCTACGGGAGGCAGCA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3). Two barcodes and six frameshifts were added to the 5 end of 338F and six frameshifts were added to the 806R primers, on the basis of a previously published protocol36. Each PCR reaction was performed in triplicate, and included a unique mixture of three frameshifted primer combinations for each plate. PCR conditions were as follows: 5l Kapa Enhancer, 5l Kapa Buffer A, 1.25l 5M 338F, 1.25l 5M 806R, 0.375l mixed plant rRNA gene-blocking peptide nucleic acids (PNAs) (1:1 mix of 100M plastid PNA and 100M mitochondrial PNA36), 0.5l Kapa dNTPs, 0.2l Kapa Robust Taq, 8l dH2O, 5l DNA; temperature cycling: 95C for 60s; 24 cycles of 95C for 15s; 78C (PNA) for 10s; 50C for 30s; 72C for 30s; 4C until use. Following PCR clean up, using AMPure beads (Beckman Coulter), the PCR product was indexed using 96 indexed 806R primers with the Kapa HiFi Hotstart readymix with the same primers as above; temperature cycling: 95C for 60s; 9 cycles of 95C for 15s; 78C (PNA) for 10s; 60C for 30s; 72C for 35s; 4C until use. PCR products were purified using AMPure XP magnetic beads (Beckman Coulter) and quantified with a Qubit 2.0 fluorometer (Invitrogen). Amplicons were pooled in equal amounts and then diluted to 10 pM for sequencing. Sequencing was performed on an IlluminaMiSeq instrument using a 600-cycle V3 chemistry kit.
library_name: TL31