title: GSM7209618: TAM nuclei from MYC-PyMT in low protein diet; Mus musculus; RNA-Seq
library_name: GSM7209618
library_construction_protocol: Tumors were collected for immune cell isolation. TAMs were sorted based on their cell surface markers (DAPI-CD45+SiglecF-Ly6G-B220-Ly6C-F4/80+Vcam+). Nuclei were isolated with an EZ PREP kit (Sigma, Cat #NUC-101) according to the manufacturer's protocol, and intact nuclei without cell contamination were further sorted by fluorescent markers (mCherry+CD11b-SYTO9+) for downstream processes. the single nucleus suspension was loaded onto Chromium Chip B (10X Genomics PN 2000060) and GEM generation, cDNA synthesis, cDNA amplification, and library preparation proceeded using the Chromium Single Cell 3' Reagent Kit v3 (10X Genomics PN 1000075) according to the manufacturer's protocol. cDNA amplification included 11 cycles, and 53-96 ng of the material was used to prepare sequencing libraries with 10-12 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/91 run using the NovaSeq 6000 SP or S1 Reagent Kit (100 cycles) (Illumina). An average of 30 thousand paired reads was generated per cell.