GSM5454349: BD Rhapsody WTA [RAILD1-NF]; Homo sapiens; RNA-Seq
Murine and human Lungs were cut into small pieces with a razor blade and digested enzymatically using 0.25 mg/ml Liberase TM (Roche) and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO) for 60 min at 37°C. Erythrocytes and dead cells were removed by 25%/65% Percoll PLUS (Cytiva) density separation. Resulting cells were stained with Calcein AM for 5 min at 37°C, immediately chilled on ice, and filtered through 40 μm cell strainer (BD). 10000 mouse cells and 6000 human cells were loaded onto BD Rhapsody cartrige. Cell load, cell lysis, mRNA trap, cDNA synthesis, and primer digestion were performed according to the manufacture's instruction. Half of the resulted beads were subjected to cDNA amplification by TAS-Seq. Sequencing libraries from amplified cDNAs were produced by using NEB UltraII FS Kit (New England Biolabs) with some modifications. Sequencing was performed by using a Novaseq S2 and a Novaseq 6000 sequencer (Illumina) according to the manufacture's instructions. 20% PhiX spike-in was used for sequencing.