GSM8996164: 2dpi, s78; Arabidopsis thaliana; OTHER
One and two days post inoculation, Arabidopsis roots were selected for a single nematode infection using microscopy. Infected seedlings were placed in a mold filled with tissue freezing medium and blue beads were placed near the nematode head to indicate the starting point for slicing before freezing the sample on dry ice. Frozen roots were cryo-sliced into 96 sections of 20-micrometer. The slices were collected using tweezers in a pre-cooled hard-shell 96-wells plate filled with 40 µL mineral oil (M8410, Sigma-Aldrich) and 7.5 ng/µL primer containing a T7 promoter, Illumina 5'adapter, 6 bp unique molecular identifier (UMI), an 8 bp section-specific barcode and a poly-T-sequence (Schild et al. 2021). The T7 promoter in the Cel-Seq2-based primers (Hashimshony et al. 2016) facilitates in vitro transcription but is not involved in the sequencing reaction. The UMI distinguishes molecules from each other, while the barcodes contribute to the spatial aspect to the Illumina sequencing library. Primers were synthesized by Integrated DNA Technologies (Leuven, Belgium). RNA amplification, cDNA synthesis and Illumina library preparation To each tissue section, 0.1 µL of 10 mM dNTPs (Promega) and 0.2 µL (1:500.000x diluted) ERCC spike-ins were added using the Nanodrop II robot (GC Biotech). The plate was heated to 65°C for 5 minutes to release the RNA and spun down in a pre-chilled centrifuge at 20.000g for 10 seconds. Next, a mix containing 0.05 µL nuclease free water, 0.4 µL First Strand Buffer (5x), 0.2 µL DTT (0.1 M), 0.1 µL RNase OUT (40 U/ µL), and 0.1 µL SuperScript II (200 U/ µL) was prepared and 0.75 µL of this mix was added to each well using the Nanodrop II for reverse transcription before incubation for 1 h at 42°C, 5 min at 4°C and 10 min at 70°C. To generate a second strand cDNA, a mix consisting of 7 µL nuclease free water 2.5 µL Second Strand Buffer (5x), 0.25 µL dNTPs (10 mM), 0.35 µL E. coli DNA ligase, 0.09 µL E. coli DNA polymerase, and 0.09 µL E. coli RNAse H was prepared and 10 µL of the mix was added to each well, followed by a 2 h incubation step at 16°C. NucleoMag NGS Clean-up and Size Select beads (LOT 102228, Machery-Nagel) were used to purify the cDNA (slightly modified from (Schild et al. 2021)). Next, the amplified cDNA was transcribed into RNA using the MEGAScript T7 transcription kit (CAT#AM1334, Ambion), and the RNA was fragmented by incubating the sample with aRNA-fragmentation buffer at 94°C for 1.5 minutes. The RNA was then cleaned up using RNA MagClean beads (C-100, Aline Biosciences) before preparing the DNA library, following the protocol of (Schild et al. 2021). The libraries were sequenced twice on the Illumina NextSeq 2000 platform using P3 reagents (100 cycles, 240Gb) at Leiden University (The Netherlands) to achieve sufficient sequencing depth. FASTQ files from both runs, containing paired-end 100 base pair sequencing reads, were merged. Read 1 was used for transcript counting based on the unique molecular identifier (UMI) and section-specific barcode, while Read 2 contained the gene-specific data.