title: GSM8122873: Sample_11_EVA71_6h_P1; Homo sapiens; RNA-Seq
library_construction_protocol: RD cells were plated on the day before the experiment (~2x106 cells in T-25 flasks). One extra flask was used to count the cells before to ensure the cells count for MOI calculation. Inoculums of 0.3 MOI were prepared fresh using Free FBS media of the three viruses (EV-A71, EV-D68 and CV-B3 P1). After removing the media, and washing with warm PBS, the cells were incubated for 1 hour with the inoculums (rocked each 10 minutes) at 37°C, for EV-A71 and CV- B3), and 33°C for EV-D68. After 1 hour, the inoculum was removed and 5% FBS media was added. The cells were incubated into their respective temperatures (37°C and 33°C) until the time point of collection (6 hpi). Mock-infected cells were also collected at 6 hpi. After 6 hpi, the cells were submitted to the standard protocol of sample preparation proposed by 10X Genomics (Chromium Single Cell 5' Reagent Kits User Guide (v2 Chemistry Dual Index), Document Number - CG00331 - Rev E, Aug, 2022). Briefly, the media is removed, and the cells are washed with warm PBS. TrypLE is added (2mL) to dissociating the cells and 2 mL of media is added to homogenize the cells. The cells are counted and also checked about their viability, after counting them, they are centrifuged at 500 x g for 5 minutes. Then, the supernatant is removed and the cells are washed with 1x PBS with 0.04% Bovine Serum Albumin (BSA), and centrifuged again at 500 x g for 5 minutes. After that, supernatant is removed and the appropriate volume of PBS/BSA is added to achieve the concentration of cells desired (~1000 cells/uL). Count the cells again and also check about cell viability. Once the cell's concentration is achieved the cells are moved to ice. For the preparation of the cDNA and sequencing library generation, we followed the instructions from the user guide Chromium Next GEM Single Cell 5' Reagent Kit v2 (Dual index) with one modification. The modification consists of a primer spike-in approach during the RT master mix preparation step.