GSM8014749: Week 3 VSV, skull base, rep 4; Mus musculus; OTHER
RNA were extracted from the tissues using RNA pure link mini kit (Thermo Fisher) as per the manufacturer's instructions. Reverse transcription (RT) was performed in two steps: first, 1 μl barcoded reverse primer mix (10 μM per each primer), 1 μl dNTP (10 mM) and 14 μl RNA template (up to 500 ng) were mixed and heated to 65 °C for 5 min followed by an immediate transfer to ice for at least 1 min. Second, mixture 2 including 4 μl 5× First-Strand Buffer, 1 μl DTT (0.1 M), 1 μl RNaseOUT (Thermo Scientific) and 1 μl SuperScriptIII (Life Technologies) was added and incubated at 50 °C for 60 min followed by 15 min inactivation at 70 °C. cDNA was cleaned up with Agencourt AMPure XP beads and PCR amplified with V-gene multiplex primer mix (10 μM each forward primer) and 3′ universal reverse primer (10 μM) using KAPA protocol (KAPA Biosystems) and the thermal cycling conditions: 1 cycle (95 °C for 5 min); 5 cycles (98 °C for 5 s, 72 °C for 2 min); 5 cycles (98 °C for 5 s, 65 °C for 10 s, 72 °C for 2 min); 30 cycles (98 °C for 20 s, 60 °C for 1 min, 72 °C for 2 min) and 1 step (72 °C for 7 min). Primers are listed in Supplementary Table 1 of Fitzpatrick et al 2020 PMID: 33149302 (doi.org/10.1038/s41586-020-2886-4).