GSM9197373: Canine MCT, LCM-dissected mast cells; Canis lupus familiaris; RNA-Seq
A separate RNA extraction step is omitted. The LCM-FFPEseq protocol was used for library preparation. 12 µL of lysis buffer was added to each 0.2 mL adhesive cap PCR tube, containing 5% PEG 8000 (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich), 0.125 µM LCM-FFPEseq-specific oligo-dT primer (5'-Biotin-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3', IDT), 0.5 mM dNTPs/each (Thermo Fisher Scientific), 0.5 U µL?¹ Recombinant RNase Inhibitor (Takara) and 0.025 µg µL?¹ Proteinase K (NEB). Volumes of PEG 8000, the oligo-dT primer, and dNTPs were adjusted to the RT volume, consistent with SS3X protocol specifications. After dispensing, the PCR tubes were centrifuged and subjected to a denaturation step of 50 minutes at 52°C followed by a proteinase K–heat inactivation step of 10 minutes at 72°C, and a hold at 4°C. Immediately following denaturation, 4 µL of the RT mix was dispensed to the tubes, composed of 25 mM TrisHCl pH 8.4 (Alfa Aesar), 30 mM NaCl (Thermo Fisher Scientific), 2.5 mM MgCl2 (Thermo Fisher Scientific), 8 mM DTT (Thermo Fisher Scientific), 1.0 mM GTP (Thermo Fisher Scientific), 0.75 µM SS3X TSO (5'-Biotin—AGAGACAGATTGCGCAATGNNNNNNNNWWrGrGrG-3', IDT), 0.5 U µL?¹ Recombinant RNase inhibitor (Takara), 2 U µL?¹ Maxima H Minus reverse transcriptase (Thermo Fisher Scientific), and 0.05 mM Proteinase K inhibitor (Sigma-Aldrich). The tubes were briefly centrifuged after RT mix dispensing. Reverse transcription was performed at 42°C for 90 minutes, followed by ten cycles of 50°C for 2 minutes and 42°C for 2 minutes, then 5 minutes at 85°C, and a hold at 4°C. Next, a PCR pre-amplification was conducted, with the addition of 24 µL PCR mix to each tube. The PCR mix consisted of 1× SeqAmp buffer (Takara), 0.025 U µL?¹ SeqAmp Polymerase (Takara), 0.5 µM SS3X forward primer (5'-CTACACGACGCTCTTCCGATCT-3', IDT) and 0.1 µM LCM-FFPEseq-specific reverse primer (5'-GTCTCGTGGGCTCGGAGATGT-3', IDT). PCR pre-amplification was performed as follows: initial denaturation at 95°C for 1 minute, followed by 19 cycles of 98°C for 10 seconds, 65°C for 30 seconds and 68°C for 4 minutes. The final elongation step was conducted at 72°C for 10 minutes. The number of PCR cycles for pre-amplification can be adapted based on the input and should be determined for each project. Subsequently, all tubes were transferred to a 96-well plate and purified using AMPure XP beads at a 0.7× ratio. Elution was performed in 16 µL of nuclease-free H2O. Quality and quantity of the cDNA libraries were checked with the Fragment Analyzer (Agilent) using the High Sensitivity NGS Fragment Analysis kit (1 bp – 6000 bp, Agilent), and a separate concentration measurement was performed using the Qubit™ dsDNA Quantification Assay Kit (Invitrogen) following the manufacturer's protocols. 1 µL of cDNA was transferred to a new 96-well plate for sample index PCR. To each well of this 96-well plate, 15 µL of Sample Index PCR mix was added, comprising 1× Phusion HF Buffer (Thermo Fisher Scientific), 0.2 mM dNTPs/each (Thermo Fisher Scientific), and 0.010 U µL?¹ Phusion HF Polymerase (Thermo Fisher Scientific). Finally, 11 µL of 0.2 µM custom premixed Nextera S50X/N70 index primers, each containing 10-bp dual indexes, were added to achieve a final reaction volume of 27 µL per well. The plate was incubated for 30 seconds at 98°C, followed by 10 cycles of 10 seconds at 98°C, 30 seconds at 55°C, and 1 minute at 72°C. The final elongation step was conducted at 72°C for 5 minutes. Purification of the final cDNA libraries was performed using AMPure XP beads at a 0.7× ratio with elution in 27 µL nuclease-free water. A strict size selection was performed to remove adaptors from the final RNA-seq libraries. cDNA fragments shorter than 220 bp were removed using E-GEL 2% (Thermo Fisher Scientific). The ZYMOclean™ Gel DNA Recovery kit (ZYMO) was then used to purify cDNA from the agarose gel with final elution in 17 µL nuclease-free water. The concentrations of the libraries were quantified using the qPCR-based KAPA Library Quantification kit (qPCR) to enable equimolar pooling.