title: GSM8486290: snmCT_jun2022_mbd56_P4; Arabidopsis thaliana; OTHER
library_construction_protocol: Nuclei extraction from pollen was performed as in Ichino et al. 2022 Cell Rep. We harvested ~ 5 mL unopened flower buds + 1 open flower per inflorescence, and ground buds in 0.1M mannitol in a mortar on ice by gently tapping. The suspension was transferred to a 50mL conical tube and vortexed intermittently for 30 sec to release spores, then filtered through a 100 um nylon membrane, then two 60 um membranes. Samples were spun 10 min 900 g at 4C with slow acceleration and braking. Pellets were resuspended in ice-cold 0.1 M mannitol, and layered above 3 mL 20% Percoll diluted in 0.1M mannitol, then centrifuged 450 g 10 min 4C. This was repeated twice. Spores were pelleted 5 min 500 g 4C, and resuspended in 800 uL Glbraith buffer + bMe. This mixture was transferred to 1.5 mL tubes containing 100 uL acid-washed 0.5 mm glass beads (Sigma), and vortexed max speed 2 min total in cold, alternating 7 sec vortex, 3 sec invert for 1st min, then 7s vortex, 2s invert for second min. Nuclei were filtered through a 10um nylon filter. Nuclei were spun 5 min 500 g 4C, and pellets were gently resuspended in Sysmex nuclei extraction buffer and DAPI buffer (1:8 ratio). Nuclei were filtered through a 35 um nylon mesh into a FACS tube and immediately sorted, using DAPI to detect nuclei. Individual nuclei were sorted into a 384 well plate pre-filled with the buffer for reverse transcription (see library construction protocol below). Each 384 well plate was processed separately, with 8 plates prepared per sample (note plate 8 of Col rep 2 and 1 of mbd56 rep 2 failed and were not sequenced). Library preparation was performed as described in Luo et al. 2022 Cell Genomics, omitting the accessibility portion of the assay (the GpC methyltransferase treatment). Briefly, RNA was first reverse-transcribed using a template-switching reaction (see SMART-seq protocols) with an oligo-dT primer in the presence of 5'-methyl-dCTP, so that all cytosines in cDNA are methylated. cDNA amplification was performed using KAPA2G DNA polymerase for TODO: TBD cycles. DNA was digested into smaller fragments using Uracil DNA gycosylase + Endonuclease VIII. then bisulfite converted using the Zymo EZ-96 DNA methylation-direct kit with reduced volume. single cell multiomics