NextSeq 500 sequencing; RNA-seq of Bur, Col and taf4b Arabidopsis thaliana meiocytes
RNA-seq of Bur, Col and taf4b Arabidopsis thaliana meiocytes
Arabidopsis thaliana stage 9 flower buds were collected and squeezed between a glass slide and coverslip to release meiocytes in prophase I (Walker et al, 2018 Nat. Genet.). Clean meiocytes free from somatic-cell debris were transferred to a new slide, washed with 1×PBS buffer and frozen in liquid nitrogen. ~2,000 meiotic cells were collected for each replicate.  Arabidopsis thaliana plants were grown under long day 16/8 hour light/dark photoperiods in a growth chamber at 21 °C and 70% humidity. Cells were lysed with lysis buffer (0.2% triton-X100 with RNase inhibitor), followed by reverse transcription using oligo-dT30VN (Sigma-Aldrich) and Superscript IV (Thermofisher).  cDNA was amplified using KAPA HiFi HotStart ReadyMix (ROCHE DIAGNOSTICS LTD) for 14 cycles before purification using Agencourt AMPure beads (Beckman Coulter). Pre-amplified libraries were quantified and quality controlled by Bioanlayzer using the High Sensitivity DNA Kit (Agilent, US). PCR amplicons were constructed into dual-indexed libraries using a Nextera XT kit (Illumina).