GSM9613938: mouse-PLN,OD3_1; Mus musculus; RNA-Seq
Total RNA was isolated from mouse tumor and lymph node tissues (including popliteal and inguinal lymph nodes) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, tissues were homogenized in 1 mL of TRIzol reagent, followed by chloroform phase separation. RNA was precipitated from the aqueous phase with isopropanol, washed with 75% ethanol, and then dissolved in RNase-free water. 1.Library Construction and Quality Control.Conventional Library Construction:mRNA was enriched from total RNA using Oligo(dT) magnetic beads. Following fragmentation, first-strand cDNA was synthesized using random hexamer primers, followed by second-strand cDNA synthesis. After end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification, the library was constructed. Library quantification was performed using a Qubit fluorometer and real-time quantitative PCR (qPCR), and the fragment size distribution was assessed using a Bioanalyzer. Strand-Specific Library Construction:mRNA was enriched from total RNA using Oligo(dT) magnetic beads. Following fragmentation, first-strand cDNA was synthesized using random hexamer primers. Subsequently, second-strand cDNA was synthesized with dUTP substituting for dTTP. After end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification, the directional library was constructed.Library quantification was performed using a Qubit fluorometer and real-time quantitative PCR (qPCR), and the fragment size distribution was assessed using a Bioanalyzer.