title: GSM6297486: HS + 0.05 µM TSA (sorted with FACS)_rep4; Brassica napus; RNA-Seq
library_construction_protocol: Prior to FACS, the microspore density was increased to 2.0 × 106 - 4.0 × 106 microspores/ml. Cultures were then filtered through 70-µm cell strainers to remove cell debris and kept on ice prior to FACS. FACS was performed with the FACSAria II (BD Biosciences, San Jose, CA, USA) flow cytometer with a 100 µm nozzle. Cells were sorted in PBS (pH 7.4) as sheath fluid at an event rate of ~5000 to 10,000 events per second and a fluid pressure of 20 p.s.i. Upon excitation at 488 nm, GFP-expressing embryogenic structures were selected based on fluorescence emission in the green channel (530 nm with a band width of 30 nm) compared with wild-type negative controls. In the corresponding FACSDiva™ software, the emission signal of populations was visualized by PE-TEXAS RED-A set against GFP+YFP-A in a dot plot. An overlay was set before sorting with the wild-type line as a negative control to set GFP negative (P1 and P3) and GFP positive (P1) fields in dot plot for sort layout. We sorted both the GFP negative population of cells (pollen and dead cells) and the embryogenic population of cells. However, due to low RNA yields obtained from the GFP negative populations, we only sorted the GFP positive population of embryogenic cells directly into RNA Extraction Buffer (10,000 cells/100 uL buffer) of the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher) and these cells were flash frozen instantly in liquid N2 and stored at -80°C for no longer than one month. RNA was purified using the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher). For RNA isolation, FACS-sorted GFP positive samples (from LEC1:LEC1-GFP cultures treated with either HS + 0.05 μM TSA or HS + 0.5 μM TSA) were pooled over one column to obtain a total of 150,000 sorted cells per biological replicate. We collected four biological replicates for the HS + 0.05 μM TSA treatment and three biological replicates for the HS + 0.5 μM TSA treatment. In addition to the FACS-sorted samples, we collected three replicates of 150,000 pollen each from non-sorted pollen cultures, as described above. For each sample, ~10 ng of RNA was converted to cDNA and amplified for 12 cycles using SMART-seq v4 Ultra Low input RNA kit for sequencing (Takara Bio USA, Inc.) according to manufacturer's instructions. Amplified cDNA was then purified by immobilization on AMPure XP beads (Beckman Coulter) and the cDNA concentration was measured with the Qubit Fluorometer and the dsDNA HS Assay Kit. The cDNA size distribution was checked using the 2100 Bioanalyzer and high sensitivity DNA kit following the manufacturer's directions. The Nextera XT DNA Library Preparation Kit and the Nextera Index Kit (Illumina, 24 indexes, 96 samples, FC-131-1001) were used to prepare cDNA libraries according to the manufacturer's instructions. As input, we used 0.125 ng amplified cDNA for the tagmentation reaction and we applied 12 cycles for the enrichment PCR of adapter-ligated fragments. To clean up the cDNA libraries, we used a ratio of 0.6:1 of AMPure XP beads (Beckman Coulter). The cDNA library quality was checked as described above (typical libraries showed a broad size distribution of ~250-1000 bp). When needed, we used a second round of purification using a 0.8: 1 ratio with SPRIselect beads (Beckman Coulter) for size selection. Libraries were diluted and sequenced with paired-end 150 base mode on a Novaseq S4 (Novogene Corporation Inc.).