title: GSM8672926: Hybrid poplar stem under severe drought condition replicate 1 bulk RNA-seq; Populus tremula x Populus alba; RNA-Seq
library_construction_protocol: To perform nuclei isolation for each library, internode 15 from one plant were placed on a glass plate with 400 μl of the Nuclei Extraction Buffer of the CyStain UV Precise P kit (Sysmex) supplemented with 2mM DTT, 0.8 U/μl of SUPERase-IN (Invitrogen), and 1:100 Protease Inhibitor (Sigma-Aldrich). Next, samples were chopped with a sterile razor blade for 2 min. This step was repeated with a 30-second interval. The homogenate was washed with 5 ml of the Staining Buffer of the CyStain UV Precise P kit from the glass plate and filtered through a pre-wetted (using the Staining Buffer) 40 μm strainer laid on top of a 50 ml conical tube placed on ice. To minimize the contamination of organelles such as chloroplast and remove the debris, the final solution, with around 5 ml of volume, was used to sort the nuclei using fluorescence-activated nuclei sorting. 80,000 nuclei were sorted with a total recovery volume of 100-120 μl in 1.5 ml RNase-free non-stick Eppendorf low binding tubes containing 10 μl of supplemented Nuclei Extraction Buffer as described above. Total RNA was extracted from hybrid poplar stem following a previosly described procedure (10.1371/journal.pone.0003567). Nuclei were loaded to the GEXSCOPE Single Nucleis RNA chip of Singleron following their procedure targetting transcriptomic data from 10,000 nuclei per library. Bulk RNA-seq libraries and sequencing was performed with the company Macrogen. Libraries were performed using the TruSeq Stranded mRNA Library Prep kit.