title: GSM9568717: Patient_PROS505-3_replicaC1; Homo sapiens; RNA-Seq
library_name: GSM9568717
library_construction_protocol: Tumor and adjacent normal liver tissues were dissociated by mincing tissue in 1-2mm squares, followed by incubation in Liver Perfusion Medium (ThermoFisher Scientific) for 15 min at 37°C in a rotating oven. After being washed in PBS (ThermoFisher Scientific), tissues were incubated with Liver Digest Medium (ThermoFisher Scientific) supplemented with HEPES and collagenase type IV (Worthington Biochemical, Lakewood, NJ) (600 to 800 U/ml) for 30 min at 37°C with rotation. Further dissociation was achieved by pipetting 10 times through a 25 mL serological pipette. Single cells were then separated from clumps using a 70 µm strainer (Fisher Scientific, Hampton, NH). After lysis of red blood cells with ACK RBC Lysis Buffer (Fisher Scientific), single cells were counted using a LUNA™ Automated Cell Counter (Logos biosystems, South Korea) and processed for scRNA-seq analysis. Patient derived spheroids embedded in Matrigel were washed with PBS, then digested in TrypLE 1X (ThermoFisher Scientific) for 30 to 45 min at 37°C with rotation to obtain a single cell suspension. Cells were washed, then, filtered through a 70-µm strainer, counted using a LUNA™ Automated Cell Counter, and processed for scRNA-seq analysis. Libraries were prepared for each pool, using 800-1000 pg of DNA and the Nextera XT DNA Library Preparation Kit. They were dual-indexed with N700 and N500 oligonucleotides. Library products were purified using SPRI beads, first at 0.6x and then at a 1x volumetric ratio. Libraries were then run on an HSD1000 tape (Agilent, Cat: 50675584) to determine the concentration between 100-1000 bp. For each library, 3 nM dilutions were prepared. These dilutions were pooled for sequencing on a NovaSeq S4 flow cell. 36 localized prostate cancer patients and 2 metastatic prostate cancer patients (one liver metastatic and one sphenoid metastatic) enrolled in this study. cDNA library preparation was performed using the Seq-Well platform as previously described. Briefly, 10,000-20,000 cells were loaded onto a Seq-Well array containing 110,000 barcoded mRNA capture beads (ChemGenes, Ct: MACOSKO-2011-10(V+)). Arrays were sealed using a polycarbonate membrane (Sterlitech, Cat: PCT00162X22100) at 37 °C for 40 minutes. Cells were then lysed in lysis buffer (5 M guanidine thiocyanate, 1 mM EDTA, 0.5% sarkosyl, 1% BME) for 20 minutes at room temperature. Hybridization of mRNA to the beads was performed in hybridization buffer (2 M NaCL, 4% PEG8000) for 40 minutes. The beads were then collected and washed with 2 M NaCl, 3 mM MgCl2, 20 mM Tris-HCl pH 8.0, 4% PEG8000. Reverse transcription was then performed using Maxima H Minus Reverse Transcriptase (ThermoFisher, Cat: EP0753) in Maxima RT buffer, PEG8000, template switch Oligo dNTPs (NEB, Cat: No447L), and RNase inhibitor (Life Technologies, Cat: AM2696) at room temperature for 15 minutes and then 52 °C overnight. Second strand synthesis was performed using Klenow Exo- (NEB, Cat: M0212L) in Maxima RT buffer, PEG8000, dNTPs, and dN-SMRT oligo for 1 hr at 37 °C.  Whole transcriptome amplification was performed with KAPA HiFi Hotstart Readymix PCR kit (Kapa Biosystems, Cat: KK2602) and SMART PCR Primer. The reactions were purified using SPRI beads (Beckman Coulter) at 0.6X and then 0.8X volumetric ratio. Libraries were prepared using 800-1000 pg of DNA and the Nextera DNA Library Preparation Kit. Dual-indexing was performed using N700 and N500 oligonucleotides. Library products were purified using SPRI beads at 0.6X and then 1X volumetric ratio. A final 3 nM dilution was prepared for each library and sequenced on an Illumina NovaSeq S4 flow cell.