Illumina MARS-Seq Emiliania huxleyi RCC6946 time course of infection with EhVM1, T3, 3dpi, control replicate 2
RNA was extracted from cell pellets using the RNeasy Plant Mini Kit (Qiagen) and was treated twice with Turbo DNase (Thermo Fisher Scientific, Invitrogen). RNA concentration and integrity were assessed using Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and TapeStation assay (Agilent Genomics). cDNA libraries were prepared according to the bulk MARS-seq protocol (Keren-Shaul et al., 2019). Starting material of 50 ng clean RNA from each sample was reverse transcribed to cDNA using oligo(dT) adaptors which contain molecular barcodes. The tagged cDNA was pooled together, converted into double-stranded DNA, and linearly amplified using T7 RNA polymerase in vitro transcription (IVT). The amplified RNA was fragmented to 200-500 bp, ligated to partial Illumina primers, and reverse transcribed to cDNA before final amplification. The final cDNA libraries were sequenced on an Illumina NovaSeq 6000 SP platform (100 cycles).