GSM8820812: poplar stem rep 1; Populus trichocarpa; RNA-Seq
To perform nuclei isolation, we followed the protocol that we previously developed for extraction of high quality nuclei from plant tissues (https://doi.org/10.1371/journal.pone.0251149). 20k nuclei were loaded to the 10X Genomics Chromium to generate the snRNA-seq library following the 10X Genomics Chromium Single Cell v3.1 protocol. The cDNA reaction was prepared as described previously (https://doi.org/10.1371/journal.pone.0251149). Prior to full-scale sample sequencing, a preliminary run was performed in an Illumina iSeq (~ 4M reads) to allow the evaluation of parameters that are indicative of the quality of the single-cell RNA-seq library, including the estimated number of cells captured and the fraction of reads in cells (i.e., percentage of ambient or "leaked" RNA). The sequencing was performed using a standard program as follows: 28 bp (Cell barcode and UMI) for read 1, 90 bp (cDNA) for read 2, 10 bp for I7 Index, and 10 bp for I5 Index. The assessment of RNA leakage was derived from the profile of the relationship between UMI counts and cell barcodes detected. If the library was considered suitable for full-scale sequencing (reads in cells > 50% and at least 3,000 cells captured at low sequencing depth) we proceeded to sequencing in an Illumina NovaSeq6000. For full-scale sequencing we targeted 150-200M reads mapped to the genome per sample, which in our experience resulted in a sequencing saturation >70% and an average of 43,000 reads per nucleus. The 10x Genomics Single Cell v3.1 Dual Index Gene Expression protocol was followed