title: GSM7159185: Neoadjuvant-P03_C; Homo sapiens; RNA-Seq
library_construction_protocol: Surgically resected fresh tumor tissue from six patients treated with two cycles of NAPC and one patient treated with two cycles of NAC was cut into pieces of approximately 1-3 mm³ (Clinicopathological information, cell counts and response to neoadjuvant therapy for single cell sequencing patients are shown in supplementary Table 1). Then, the pieces were transferred to the gentle MACS C Tubes (Miltenyi Biotec, Bergisch Gladbach, Germany), with 5 mL of digestive enzyme included in Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Then the tissues were made into single-cell suspension using the gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) for 60 min on a rotor at 37℃ according to the manufacturer's instructions. After removing of red blood cells using erythrocyte lysis solution (Miltenyi Biotec, Bergisch Gladbach, Germany), the cells were filtered using a 40 µm strainer (BD Falcon, New York, USA). CD45+ cells were sorted by using the Human CD45 Microbeads Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell suspensions (700-1200 living cells/μL determined by trypan blue staining) were loaded on a 10x Genomics ChromiumTM Single Cell Controller Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell gel beads in emulsions (GEMs) by using Chromium Single Cell 5' Library & Gel Bead Kit v1.0 (10x Genomics, Cat# 1000006) and Single Cell A Chip Kit (10x Genomics, Cat# 1000152). Captured cells were lysed and the released RNA was barcoded through reverse transcription in individual GEM. Barcoded cDNAs were pooled and cleanup by using DynaBeads MyOne Silane Beads (Thermo Fisher, Cat# 37002D), and then amplified and cleanup for further next generation library construction.