GSM4942793: CTCL_P4_PRE_B_2; Homo sapiens; RNA-Seq
Two serial sections of the tissue microarray were taken at 7 µm thickness and mounted onto frame slides with polyethylene naphthalate membranes (Thermo Fisher Scientific, #LCM0521). Slides were immersed for 20 s each in xylene (three times), 100% ethanol (three times), 95% ethanol (two times), 70% ethanol (two times), water, hematoxylin (Dako, #S3309), water, bluing reagent (Thermo Fisher Scientific, #7301), water, 70% ethanol (two times), 95% ethanol (two times), 100% ethanol (three times), and xylene (three times). Immediately after staining, cells were dissected from every tissue microarray spot on an ArcturusXT LCM System (Thermo Fisher Scientific) using the ultraviolet laser to cut out the desired region and the infrared laser to adhere the membrane to a CapSure HS LCM Cap (Thermo Fisher Scientific, #LCM0215). A tissue area containing roughly 1000 mononuclear cells was captured from each spot, with cell numbers determined based on density estimates by cell counting in an adjacent H&E-stained section. If a core had more than 1000 mononuclear cells, a tissue fragment containing around 1000 mononuclear cells was dissected from that core. If a core had less than 1000 cells, tissue fragments from corresponding cores on the serial section membrane were combined in the same LCM cap to obtain approximately 1000 cells. After microdissection, the caps were sealed using 0.5-ml tubes (Thermo Fisher Scientific, #N8010611) and stored at -80 °C until cDNA library preparation. Sequencing libraries were prepared according to the Smart-3SEQ protocol for LCM HS caps as previously described with slight modifications21. Briefly, 10 µl of lysis mix consisting of 40% (v/v) 5 M trimethylglycine solution (Sigma, #B0300), 20% (v/v) 10 mM nuclease-free dNTP mix (Thermo Fisher Scientific, #R0192), 10% (v/v) 20 µM first-strand primer in TE buffer (1S, /5Biosg/GT GAC TGG AGT TCA GAC GTG TGC TCT TCC GAT CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TV; Integrated DNA Technologies), 10% (v/v) Triton-X 100 (Sigma #T8787; diluted to 0.5% v/v in molecular biology-grade water), and 20% (v/v) Proteinase K (New England Biolabs, #P8107S; diluted to 0.125 mg/ml in in molecular biology-grade water) was added to the center of each LCM cap. Caps were sealed with 0.2 ml low-retention PCR tubes (Corning, #PCR-02-L-C) and incubated on a pre-warmed metal CapSure incubation block (Thermo Fisher Scientific, #LCM0505) at 60 °C in an incubator. Then, tubes were briefly centrifuged, and 10 µl of template-switching reverse-transcription (TS-RT) FFPE LCM mix consisting of 40% (v/v) 5x SMARTScribe first-strand reaction buffer (Clontech, #639537), 20% (v/v) 20 mM DTT (Clontech, #639537), 10% (v/v) 20x RNase inhibitor (Thermo Fisher Scientific, #AM2694), 4% (v/v) 50 µM second-strand primer in TE buffer (2S, /5Biosg/CT ACA CGA CGC TCT TCC GAT CTN NNN NrGrG rG; Integrated DNA Technologies), 4% (v/v) 200 mM MgCl2 (Sigma, #63069), 2% 5 mM proteinase K inhibitor (EMD Millipore, #539470), and 20% (v/v) 100 U/µl SMARTScribe reverse transcriptase (Clontech, #639537) was added. Samples were incubated in a programmable thermal cycler (42 °C for 30 min, 70 °C for 10 min, 4 °C hold), and 1.25 µl of a unique P5 primer and 1.25 µl of a universal P7 primer (2 µM in TE buffer each; Integrated DNA Technologies; sequences available upon request) and HiFi HotStart ReadyMix (Kapa, #KK2601) were then added, followed by 22 cycles of PCR amplification (98 °C for 45 s; 22 cycles at 98 °C for 15 s, 60 °C for 30 s, 72 °C for 10 s; then 72 °C for 60 s, and 4 °C hold). Amplified cDNA was next purified with SPRI bead mix (Beckman Coulter, #B23317) and a magnetic separation block (V&P Scientific, #VP772F4). Finally, the samples were washed with 80% ethanol and resuspended in TE buffer to yield the sequencing-ready library.
GEO Accession
GSM4942793