GSM8252934: control untreated replicate 2; Phaeodactylum tricornutum; OTHER
20,000 cells were sorted per sample. Samples were sorted directly into cold lysis buffer, in Eppendorf™ LoBind tubes that were kept on ice prior to sorting and during sorting at 4°C, and immediately after sorting underwent vortex, spin down, and flash frozen using liquid N2. Samples were kept at -80°C until further processing. RNA extraction was conducted using the NucleoSpin® RNA XS kit (Machery-Nagel, Düren, Germany). cDNA library preparation was done using the MARS-seq protocol as described in Keren-Shaul et al., 2019 (https://doi.org/10.1038/s41596-019-0164-4), adapted for small amount bulk RNA samples. Following RNA extraction, reverse transcription (RT) was done to generate barcoded cDNA containing the unique molecular identifier (UMI) sequences (unique to each mRNA molecule) and the sample ID barcodes. Unused primers were then degraded with Exonuclease I (NEB). The cDNA from different samples was then combined into pools, and underwent second-strand synthesis and in vitro transcription to amplify the sequences linearly. RNA products were fragmented and ligated with single-stranded DNA adapters containing pool-specific barcodes. A second round of RT was carried out, followed by polymerase chain reaction with primers containing Illumina adapters to construct DNA libraries for paired-end sequencing on a NextSeq machine (Illumina).