title: GSM5415344: VMH from 5H7 [LID_105140]; Macaca mulatta; RNA-Seq
library_construction_protocol: Total RNA was extracted using a TRIzol-chloroform gradient followed by column isolation of RNA using the Quick-RNA Microprep kit (Zymo Research). Briefly, 1 ml of TRIzol (Invitrogen) was added to each 2-ml sample tube, along with a sterile 5-mm stainless steel grinding bead (QIAGEN cat. no. 69989). Samples were then homogenized using the Tissuelyser II system (QIAGEN) at 20 Hz in two sets of 2 minutes each, with rotation in between, and the lysate transferred to a new tube. 200 µl of chloroform were added to each sample, which was then vortexed at full speed for 15 seconds and incubated at room temperature for 2 minutes. The sample was then centrifuged at 12,000 × g for 15 minutes at 4°C. After centrifugation, the aqueous layer was transferred to a new tube and mixed with 600 µl of 70% ethanol to precipitate RNA. The resulting solution was transferred to Quick-RNA Microprep columns (Zymo Research). The remainder of the protocol followed manufacturer instructions for the Quick-RNA Microprep kit. Total RNA was eluted in 60 µl of RNase-free water. We prepared bulk RNA-seq libraries using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) with an initial mRNA isolation step using the Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). We followed manufacturer instructions and guidelines for library preparation using these kits. We used 200 ng of starting RNA and set the enzymatic fragmentation time (10 minutes), first-strand synthesis incubation time (50 minutes at 42°C), and bead cleanup volume ratios for a target insert size of 400 bp. Libraries were amplified using 10 cycles of PCR and eluted in a final volume of 20 µl of RNase-free water. Libraries were quantified by Qubit 3 using the Qubit DNA HS Assay kit (Invitrogen) and library quality was assessed using the Fragment Analyzer HS NGS kit (AATI/Agilent). Libraries were pooled in equimolar quantities prior to sequencing.