title: GSM3323514: GM182_6 RNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: 5 µm serial sections of FFPE tissue were stained with H&E and Aniline blue. H&E stained slides were used to confirm histology of tumors and guide bordering consecutive serial sections of Aniline blue stained tissue samples. Bordered tissue samples were microdissected using sterile disposable scalpels and razor blades with the assistance of a dissection scope. 10 – 14 pieces of each sample isolated from serial sections were pooled in a microcentrifuge tube containing 100% ethanol. RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Thermo Fisher Cat#AM1975) was used to isolate RNA from FFPE tissues according to the manufacturer's protocol. RNA samples were quantified using NanoDrop 8000 and Qubit RNA HS Assay. RNA integrity numbers were calculated on Agilent HS RNA ScreenTape. RNA was subjected to library construction with the Illumina TruSeq Stranded mRNA Sample Preparation Kit (Cat#RS-122-2101, RS-122-2102) according to manufacturer's protocol. Chemically denatured sequencing libraries (25 pM) are applied to an Illumina HiSeq v4 single read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (Cat#GD-401-4001). Following the transfer of the flowcell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50-cycle single-read or 125-cycle paired-end sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (Cat#FC-401-4002).
attributes.tag: GEO Accession
attributes.value: GSM3323514