HSEB-Middle 2 of 3 replicates, Control, collected 2022-05-05
DNA from pollen samples was isolated from collected pollen samples using the Qiagen DNeasy Plant Pro Kit. DNA samples were individually amplified through PCR, targeting the trnL (UAA) c-d chloroplast intronic region, with forward primer 5-CGAAATCGGTAGACGCTACG-3 and reverse primer 5-GGGGATAGAGGGACTTGAAC-3. To allow for multiplexing, primers were modified by attaching unique 24 base pair indexes to four forward and reverse oligo primers. DNA libraries were prepared using an in-house pipeline in accordance with ONT protocols. Library preparation first consisted of end-repair and A-tailing reactions using NEBNext Companion module for ONT Ligation Sequencing. Adapter ligation and loading preparation were performed using ONT Ligation Sequencing Kit V13, SQK-LSK110. Pool 1 was processed to be sequenced on a MinION Flow Cell (R9.4). Pool 2 was processed to be sequenced on the MinION Flongle Flow Cell (R9.4). Sequencing was performed on the ONT MinION Mk1C device. Experiments were run for 48 hours or until pore exhaustion.