GSM9428981: WNV, 6dpi, 1; Mus musculus; RNA-Seq
Mid and distal regions of small intestines (10-cm tissue measured from cecum) were cut into 5-cm segments, flushed with PBS, and positioned on a glass rod. After creating a shallow incision with fine forceps, the muscularis externa was separated by gently rolling it away with a cotton swab. The muscularis externa and the remaining gut tissue were minced using scissors (0.5 mm pieces) and digested in buffer (RPMI 1640 with 20 mM HEPES (Gibco, # 24020117), 400 U/mL collagenase D, 2.5 U/mL Liberase TM (Roche, # 05401127001), 50 µg/mL DNase I (Roche, # 10104159001) at 37°C on a shaker (140 rpm) for 40 min. Digested tissue was further dissociated using a 1-ml pipette, After filtering through a 70-µm strainer and washing with FACS buffer (PBS supplemented with 2% FBS, 5 mM EDTA, 0.1% NaN3). Cells were sorted using Fluorescent Activated Cell sorting directly into extraction buffer using MACSQuant Tyto Cartridges and a MACSQuant Tyto sorter (ThermoFisher/Applied Biosystems, #12204-01). Library preparation was performed with 1-50 ng of total RNA.  cDNA was prepared using the SeqPlex RNA Amplification Kit (Sigma) per manufacturer's protocol and then fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, an A base was added to the 3' ends, and then Illumina sequencing adapters were ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.