GSM9694620: MiaPaca_CAMtumour_LaNta31_9; Gallus gallus; Homo sapiens; RNA-Seq
CAM tumour tissues were dissected then fixed in 10% neutral buffered formalin for 24 h and subsequently transferred to RNase-free water and stored at 4°C until RNA isolation. Samples were homogenised using a sonicator, and total RNA was extracted using the Monarch Total RNA Miniprep Kit (New England Biolabs), following the manufacturer's instructions. Initial quality control was performed using a NanoDrop spectrophotometer to confirm RNA presence, then using the High Sensitivity RNA ScreenTape Analysis Kit (Agilent Technologies, 5067-5577). For this, 1 µL of each RNA sample or a diluted RNA ladder (Agilent Technologies, 5067-5581) was mixed with 5 µL of High Sensitivity RNA Sample Buffer (Agilent Technologies, 5067-5580), vortexed at 2000 rpm for 1 minute, denatured at 72 °C for 3 min, and cooled on ice. Samples were analysed using the Agilent 2200 TapeStation Instrument, and only those with RNA Integrity Number equivalent (RINe) values between 8 and 10 and 28S:18S rRNA ratios greater than 2 were selected for sequencing. mRNA was purified using poly-T oligo-attached magnetic beads, fragmented using divalent cations under elevated temperature, and reverse transcribed using random hexamer primers and M-MuLV Reverse Transcriptase. Second-strand synthesis was performed using DNA Polymerase I and RNase H, and resulting cDNA fragments were end-repaired, adenylated, and ligated to adaptors with hairpin loop structures. Fragments of 370–420 bp were selected using the AMPure XP system (Beckman Coulter), and libraries were amplified using Phusion High-Fidelity DNA polymerase with indexed primers. Library quality was assessed using the Agilent Bioanalyzer 2100 system, and clustering was performed on a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina).
GEO
GSM9694620_r1