title: GSM9327422: PDAC biopsies; Patients 2, 3, 4, 6, 8 ; C4D1, Cycle 12, baseline [combined]; Homo sapiens; RNA-Seq
library_construction_protocol: STc buffer (146 mM NaCl, 20 mM Tricine, 1 mM CaCl2, and 21 mM MgCl2) and NSTcPA buffer (146 mM NaCl, 20 mM Tricine, 1 mM CaCl2, and 21 mM MgCl2, 0.2% NP-40, 0.01% BSA, 0.15 mM Spermine, 0.5 mM Spermidine) were prepared fresh. Tumor biopsies were lysed and nuclei isolated on a per-sample basis. Frozen tissues were then transferred into 500 mL of NSTcPA buffer containing DNA and RNA and minced for 8 minutes. Minced tissues were pipetted over a 40 mM filter into a 50 mL conical tube. The well was washed twice with 1 mL of NSTcPA buffer and twice with 1 mL of ST buffer and pipetted over the same filter for each subsequent wash. Subsequently, 1 mL of ST buffer was pipetted over the filter twice to further dilute the sample. The sample was then transferred to a 15 mL conical tube and centrifuged at 500 x g for 5 minutes at 4°C, with deceleration set to 5. The supernatant was removed, and the pellet was resuspended in 100-200 uL of PBS  /1% BSA, then pipetted through a 35 mM FACS tube. Nuclei were quantified using a disposable C-Chip hemocytometer, and up to 100,000 nuclei per sample were immediately fixed using the Evercode Low Input Cell Fixation kit according to manufacturer's instructions. Nuclei samples were then stored at -80°C until all samples were ready to proceed with library preparation. Sequencing library preparation with 12,500 cells per library was performed with the Evercode Whole Transcriptome v3 kit according to manufacturer's instructions. Sublibrary quality control and quantification were performed using Qubit fluorometer, Agilent 4200 TapeStation, and KAPA qPCR analysis using a Quant studio 5, and sublibraries were then pooled to 4 nM for sequencing based on qPCR results. Seven sublibraries were pooled and submitted for sequencing in total.