Chloroplast RNA Extraction from Tobacco

Chloroplasts were extracted according to “Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves” bio protocol (Klinkenberg 2014 (link); Klinkenberg et al. 2014 (link)). Briefly, fresh leaf tissue was ground, filtered and centrifuged through a Percoll gradient and visualized on an inverted microscope. Chloroplasts were then shock-frozen and total RNA was isolated from purified chloroplasts using Trizol (Thermo Fisher Scientific, Waltham, MA) or RNeasy Plant Mini kit (Qiagen, Germantown, MD) as per manufacturers’ instructions. For each plant, approximately 100 mg of tissue was ground from each leaf to isolate chloroplast RNA. Leaves from individual plants were pooled. Removal of chloroplast DNA was done by treating the samples with Ambion rDNase1 (Thermo Fisher Scientific, Waltham, MA). Because rRNA typically constitutes over 75% of total RNA and its depletion can results in very low yields of RNA for cDNA preparation, rRNA depletion was not performed. The RNA integrity of the isolated RNA was examined on a Bioanalyzer machine and quantitated on a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA) prior to library preparation. For cDNA synthesis, about one microgram of non rRNA-depleted RNA was used to make double strand cDNA (ds-cDNA) and dsDNA was produced using the Invitrogen SuperScript II Double Stranded cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA) with random hexamers primers for first-strand synthesis. The cleaned ds-cDNA was then used to construct a library using the Illumina Next Tera Library prep kit with no adaptations (Illumina, Inc, San Diego, CA). After examination of the library quality using the Bioanalyzer (Agilent, Santa Clara, CA), multiplexed libraries were sequenced using the Illumina MiSeq sequencing platform per standard MiSeq run parameters (Illumina protocol manuals).