GSM7666477: Patient 2, NSCLC tumor-draining lymph node, LN2; Homo sapiens; RNA-Seq
Lymph node tissues were obtained from the pathological specimens of LN dissection. Rapidly after resection, lymph nodes were placed in 10% neutral buffered formalin solution, fixed at room temperature and embedded in paraffin blocks (FFPE). FFPE blocks were sliced into 4 µm unstained sections on glass slides for RNA extraction. RNA was extracted using the RNeasy FFPE kit (Qiagen) following scraping of LN tissues with a razor blade from 15 glass slides per sample and de-paraffinization using the Deparaffinization Solution (Qiagen). RNA concentration was quantified by spectrophotometry (NanoDrop™ One, Thermo Scientific) and fluorometry (Qubit™, Invitrogen), and RNA quality (DV200) was assessed using the Fragment Analyzer 5200 System (Agilent) and the DNF-472 HS RNA (15nt) kit (Agilent). Total RNA libraries were prepared using Illumina stranded Total RNA prep ligation with Ribo-Zero Plus kit (Illumina) starting with an input amount of 187-200 ng total RNA. The size and quality of the libraries were evaluated with the Fragment Analyzer 5200 System and the DNF-474 HS NGS Fragment kit (1-6000bp) (Agilent). The KAPA Library Quantification kit for Illumina platforms (KAPA Biosystems, Roche) was used to quantify the libraries. Samples were pooled in equimolarity. The double indexed libraries were sequenced on a NextSeq 550 instrument (Illumina) using 74 base-length read chemistry in paired-end mode.