title: GSM8761505: Breast cancer tissue; Homo sapiens; RNA-Seq
library_construction_protocol: The tissue sample was minced into approximately 1–2 mm3 pieces, washed with phosphate-buffered saline (PBS), and placed in Accumax cell detachment solution (Merck Life Science, Milan, Italy) for 45 min at room temperature with gentle but constant mixing. After letting the remaining tissue pieces settle down (for 2 min), the supernatant was filtered to obtain a single-cell suspension. Next, cells were centrifuged (300 rcf for 5 min). After discarding the supernatant, cells were resuspended into chilled 1X DPBS and centrifuged (300 rcf for 5 min). After discarding the supernatant, chilled 100% methanol was added drop by drop to 1×10^6 cells that were stored at -80°C for scRNA-seq library construction and sequencing. The tube containing methanol-fixed cells, stored at -80°C, was equilibrated to 4°C for 5 min. Fixed cells were centrifugated at 1.000 RCF for 5 min at 4°C. After the supernatant was removed without disrupting the cell pellet, an appropriate volume of Wash-Resuspension Buffer (0.04% BSA - 1mM DTT - 0.2 U/ul RNase Inhibitor in 3X SSC Buffer) was added to obtain the desired cell concentration. Cell concentration was determined using a Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA). The Trypan Blue staining of the methanol-fixed cells showed that 100% of the cells were dead, indicating that all cells were effectively fixed and permeabilized. For the library preparation and sequencing, Chromium Controller and Chromium NextGEM Single Cell 3' Reagents Kit v3.1 (10X Genomics, Pleasanton, CA) have been used for partitioning cells into Gel Beads-in-emulsion (GEMs), where all generated cDNA shares a common 10x barcode. Libraries were generated from the cDNA following the manufacturer's instructions. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. TruSeq Read 1 (read 1 primer sequence) is added to the molecules during GEM incubation. P5, P7, a sample index, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers were used in Illumina bridge amplification. After checking with both Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent Bioanalyzer DNA assay (Agilent Technologies, Santa Clara, CA), libraries were prepared for sequencing and sequenced on paired-end 150 bp mode on NovaSeq6000 (Illumina, San Diego, CA).