Fig 1.
Workflow diagram showing the nuclei isolation and snRNA-seq library construction procedure from Populus shoots and stem.
Fig 2.
Fluorescence Activated Nuclei Sorting.
(A, B) To minimize the contamination of organelles such as chloroplast and remove the debris that could clog the 10× Genomics microfluidic chips, we sort the nuclei using FANS technology using a BD FACSAria™ IIU/III upgraded cell sorter. (C) An initial gate was drawn on a FSC and SSC plot to eliminate the majority of non-nuclear debris and organelles. The final histogram was used to isolate the desired nuclei. 40,000 DAPI+ nuclei were sorted with a total recovery volume of 67–70 μl, into a 1.5 ml RNase free non-stick Eppendorf low binding tube containing 10 μl of NIB WASH.
Table 1.
Summary of snRNA sequencing and quality control overview for each sample.
Fig 3.
Identification of true nucleus-associated barcodes.
Cumulative UMI counts plot—nucleus-associated barcodes, arranged in decreasing order (from highest to lowest UMI counts) versus the cumulative UMI counts. The vertical dotted lines indicate the 1,000 UMI-tagged transcripts cutoff. Nucleus barcodes with at least 1,000 UMIs were considered true nucleus. (A) Whole stem from biological replicate 1 and 2 (black and blue lines, respectively) of Populus trichocarpa. (B) Shoot apical meristem of Populus tremula × alba 717-1B4.