title: GSM7025751: Sunn4_24h; Medicago truncatula; RNA-Seq
library_construction_protocol: Nuclei isolation was carried out at 4. Forty root segments were moved to a glass plate with 200 μL of modified Nuclei Isolation Buffer containing 0.5 U/uL Protector Rnase Inhibitor. After samples were fragmented with a sterile razor blade, the homogenate was washed into a 50 mL tube with NIB and incubated on a shaker for 5 min. All samples went through at least two stages of filtration, first with Miracloth and then with a 40 μm cell strainer. The samples were centrifuged for 5 min at 600 g before the supernatant was discarded without disturbing the pellet. The pellet was resuspended with 4 mL of NIB WASH. Centrifugation and resuspension were repeated once more. After the final centrifugation, 300 μL of NIB WASH was used to resuspend the pellet. Samples that contained debris underwent an additional filtration step with a 40 uM cell strainer with an attached 5 mL tube. Next, the suspension was transferred to a sterile tube compatible with the BD FACSAria™️ IIU/III upgraded cell sorter. Nuclei were stained with 5 μg/mL DAPI for 5 min at 28 °C before sorting. The sample was used for Florescence Activated Nuclei Sorting (FANS) before being loaded onto the 10× Genomics microfluid chip. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.