GSM8396064: Mycosis fungoides, individual 19, rep 2; Homo sapiens; RNA-Seq
Microdissection was performed for 8–15 paraffin-embedded slide–sections sequentially sliced with a thickness of 5 µm and installed on polyethylene terephthalate slides (Leica AS LMD; Leica Microsystems Ltd., Wetzlar, Germany). The paraffin was removed by dipping the samples twice in xylene for 10 min, and thereafter the sections were stained with hematoxylin for 10 min. CD3 antibody-stained slides (NCL-L-CD3-565) were used to differentiate between lymphocytic and epithelial cells (Supplementary Figure 6). Laser capture microdissection (LCM) using a ZEISS PALM MicroBeam Microdissection System (CarlZeiss Microscopy GmbH, Oberkochen, Germany) and adhesive collection caps with a minimum microdissected lymphocytic cell area of 5 × 106 µm2 was conducted for each sample according to the recommendations of the Quiagen RNA extraction manuals. Following microdissection, the samples were stored in collection vessels containing appropriate volumes of Deparaffinization Solution (#19093, Qiagen GmbH, Hilden, Germany), and the RNA was extracted using a MiRNeasy FFPE extraction kit (#217504, Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. After adding the proteinase K solution, the samples were incubated overnight at 56 ◦C with gentle shaking to obtain a better yield of RNA. The quality and quantity of the extracted RNA samples were analyzed using a LabChip GX Touch HT RNA Assay Reagent Kit (PerkinElmer, Waltham, MA, USA), and a Qubit RNA BR Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for the genomic DNA contamination measurements. Dual-indexed mRNA libraries were prepared from 150 ng of total RNA using the QuantSeq 3' mRNA–Seq Library Prep Kit FWD (Lexogen GmbH, Vienna, Austria) according to user guide version 015UG009V0251. During second strand synthesis, 6 bp Unique Molecular Identifiers (UMI) were introduced with the UMI Second Strand Synthesis Module (Lexogen Gmbh, Vienna, Austria) for the detection and removal of PCR duplicates. Library quality was measured using a LabChip GX Touch HT DNA High Sensitivity Reagent Kit (PerkinElmer, Waltham, MA, USA). Sequencing was performed using the NovaSeq 6000 System (Illumina, San Diego, CA USA) with a read length of 2 × 101 bp and a target coverage of 10 M reads per library.