Nucleospin rna clean up xs

Total RNA was extracted from the subjects’ saliva samples with a volume ≥ 2 mL using either the RNeasy micro kit (Qiagen, Hilden, Germany) or the NucleoSpin^® RNA kit (TaKaRa Bio, Shiga, Japan) according to the manufacturers’ protocol. NucleoSpin^® RNA Clean-up XS (TaKaRa Bio) was used to purify and concentrate the diluted RNA if the amount of total RNA was insufficient for RNA sequencing (RNA-seq). The sequence library was prepared using the TruSeq^® Stranded Total RNA Library Prep kit (Illumina, San Diego, CA, United States) and was sequenced as 100-bp paired-end (PE) reads on an Illumina NovaSeq 6000 platform (Illumina). The library preparation and RNA-seq were performed by Macrogen (Seoul, South Korea).
We used the fastp ver. 0.19.7 (Chen et al., 2018 (link)) to remove the Illumina PE adapters, polyG tail (and polyX tail), and low quality reads with the following parameters: −q 15 −n 10 −t 1 −T 1 −l 20. The filtered PE reads were mapped to the human reference genome (GRCh38) with annotation information included in the gtf (gene transfer format) file from Ensembl GRCh38.96, using the STAR ver. 2.5.3 (Dobin et al., 2013 (link)) after the reference genome was indexed by the RSEM ver. 1.3.0 (Li and Dewey, 2011 (link)). The RSEM program was also used to estimate gene expression levels from the mapped BAM (binary alignment map) files.

Total RNA was extracted from cells using NucleoSpin RNA (Takara) and purified using NucleoSpin RNA Clean-up XS (Takara). cDNA was synthesized by reverse transcription using a PrimeScript RT reagent Kit (Takara). Quantitative RT-PCR for DOCK11 mRNA was performed using TB Green Premix Ex Taq II (Takara) and Thermal Cycler Dice Real Time System Lite TP700 (Takara). GAPDH mRNA was quantified and standardized in the same samples. Primer sets were as shown in Table S3.

To measure transcription and translation activities of hybrid cells, plasmid DNA or mRNA that encodes lacZ was introduced into ALBiC reactors prior to fusion with protoplast cells. mRNA encoding lacZ was prepared by using in vitro Transcription T7 Kit (Takara Bio, Inc.), and was purified using the NucleoSpin RNA Clean-up XS (Takara Bio, Inc.). The yield of the transcripts was measured by a NanoDrop 2000 Spectrophotometer (Thermo Fischer Scientific). The transcripts were freshly prepared before each assay. E. coli strain Top10 expressing mseCFP was used for protoplast preparation, as lacZ genes are knocked out in Top10. SP buffer containing 30 ng/μL of plasmid or 95 ng/μL of mRNA and 5 mM of ATP were introduced in the ALBiC reactors before fusion, resulting in approximately 2.73 × 10² molecules of plasmid or 1.44 × 10³ molecules of mRNA in each reactor. After 3 h of incubation, 150 μL of GP medium containing 100 μM spiro-based immobilisable diethylrhodol-βGal (SPiDER-βGal)^{52 (link)} was injected into the ALBiC.