8439-B16BL6
Baseline (untreated) or treated tumors (ICI or anti-CD4 Antibody) were harvested at 500~1000mm3 and were subjected to NGSmIO panel test. Total RNA was prepared from samples using RNeasy Mini Kit (QIAGEN, Cat.74106) per manufacturers instruction. The quantity and quality of the total RNA are assessed using NanoDrop 2000 and Agilent 2100 BioAnalyzer. Only high-quality RNA sample (OD260/280=1.8-2.0, OD260/2302.0, concentration> 20ng/L, amount>1g, volume>30L, RIN (RNA integrity number) 7) is used for the library construction and sequencing. PolyA mRNA was the isolated from the total RNA samples using two serial rounds of binding to oligo (dT) magnetic particles (Agilent) then chemically-fragmented to a size appropriate for RNA sequencing library preparation using RNA Seq Fragmentation Mix (Agilent). Using these short fragments as templates, first stranded cDNA was synthesized by RNA Seq First Strand Master Mix (Agilent) and Actinomycin D (Agilent), followed by purification using AMPure XP Beads (Beckman Coulter). Second-strand cDNA synthesis and end repair were then performed using RNA Seq Second Strand plus End Repair Enzyme Mix (and RNA Seq Second Strand + End Repair Oligo Mix, Agilent), followed by purification using AMPure XP Beads (Beckman Coulter). The purified cDNA was then subjected to cDNA 3 ends dA-tailing using RNA Seq dA Tailing Master Mix (Agilent) and adaptor ligation using SureSelect Ligation Master Mix (Agilent), followed by purification using AMPure XP Beads (Beckman Coulter). The adapter-ligated cDNA library was then amplified by pre-capture PCR Reaction Mix (Agilent) and purification. After cDNA library construction, Qubit 3.0 fluorometer dsDNA HS Assay (Thermo Fisher Scientific) was used to quantify concentration, while the size distribution was analyzed using Agilent BioAnalyzer 2100 (Agilent). cDNA library with good quality and concentration was then used for the sequencing library hybridization using SureSelect Block Mix (Agilent) and RNA Capture Library Hybridization Mix (Agilent). Capture of hybrids was performed by streptavidin beads (prepared from Dynabeads M-270 Streptavidin Beads (Thermo Fisher Scientific)). Sequencing libraries was cleaned up after PCR amplification of captured libraries and indexing. After library quality control (QC), MGI Easy Universal Library Conversion Kit (MGI) with High-Throughput Pair-End Sequencing Primer Kit (MGI) was used to generate clusters. Paired-end sequencing will be performed using a MGISEQ2000 system following MGI-provided protocols for 2 x 150 paired-end sequencing.