GSM8535026: AdrenocorticalTumor_ACME_scFASTseq_1; Homo sapiens; RNA-Seq
Single cells were isolated from tissue samples using ACME HS followed by cryopreservation in 3xSSC*10% DMSO and methanol cell fixation. Key adaptations of the ACME HS method were the supplement of solution composition with 0.1M NAC and the introduction of additional washing steps in cold high salt 3xSSC* buffer. ACME dissociation was conducted in ~ 1 hours on a rotator at room temperature, with periodic pipetting of the solution. Afterward, we removed the ACME solution and washed the pellet with a two-step washing in cold 3xSSC. Single cells were captured, barcoded, and cDNA libraries were generated using the V1.1-SeekOne DD Single Cell Full-length RNA Sequence Transcriptome-seq Kit. 10,000 cells in cold 3xSSC* were mixed with RT-PCR master mix and immediately loaded together with scFAST-seq Barcoded Beads and Carrier Oil into a Chip S3. The cDNA libraries were quantified using the Qubit dsDNA HS assay kit (Thermo Fisher Scientific), and the fragment sizes were assessed with the Agilent 5200 Fragment Analyzer using a DNA HS (1-6000NT) kit. The final library was sequenced on an Illumina Novaseq 6000 platform using the S4 Reagent Kit v1.5 (200 cycles).