title: GSM4140404: carcinoma_39; Mus musculus; RNA-Seq
library_construction_protocol: By combining the recently published Geo-seq protocol using SmartSeq2 for laser capture microdissected samples with a recent report for parallel DNA and RNA sequencing from single cells, representative cDNA libraries of laser-captured regions from fixed tumour sections were generated. In brief, 7 µm thick cryosections were mounted onto Menzel Superfrost Plus slides and placed in decreasing and increasing ethanol solutions. Zonal patches of clonal populations of various stages in the primary tumour as well as monochromatic metastases were microdissected by using a Zeiss Laser Capture Microscope (energy: 49, focus: 67, delta-energy: 15, delta-focus: 5; CFP (474/23), GFP (510/20), YFP (535/22) and RFP (620/60)) in Auto LPC mode. Clonal populations of normal mammary glands were pooled to obtain a sufficient sample size. Collected laser capture microdissected samples were first lysed at 37 °C for 30 minutes by using a digestion buffer containing proteinase K (150 mM NaCl, 100 mM Tris pH 7.5, 0.5% Igepal, 0.5 µg/µl proteinase K) to reverse the cross-linking of formalin fixation and for further 30 minutes at 42 °C by using a 4M GuSCN solution (Thermo Fisher). Biotinylated oligo-dT30VN beads were used to discard gDNA in the supernatant and specifically start the reverse-transcriptase reaction and cDNA preamplification on poly-adenylated mRNA (exception to GeoSeq protocol: dNTP mix contained 10mM each of dATP, dCTP, dGTP, dTTP and SmartSeq2 ISPCR oligos were used, 5′-AAGCAGTGGTATCAACGCAGAGT-3′). cDNA quality was assessed by a Qubit Fluorometer with a dsDNA HS assay kit (Thermo Fisher), an Agilent high-sensitivity DNA chip for cDNA size distribution and quantitative real-time PCR to check for the expression of housekeeping genes and the absence of gDNA. Laser capture microdissected samples with best quality were subjected to RNA sequencing. For library preparation, 500 pg cDNA was amplified using the Nextera XT DNA library preparation kit (Illumina). 12 PCR cycles were carried out, DNA was purified using AMPure XP beads in a 0.8:1 ratio and cDNA was sequenced on a NextSeq 500 platform (Illumina) with a 75-bp single-read mode (Index1:8, Index2: 8).
attributes.tag: GEO Accession
attributes.value: GSM4140404