GSM5454351: BD Rhapsody WTA [Bdofficial]; Mus musculus; RNA-Seq
The spleen was mushed on cell strainer with 5 ml of preparation medium and resulted cells from three spleen were harvested and pooled. Then, spleen cells were suspended with ACK lysis buffer [155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3] and incubated for 10 min at room temperature. The cell suspension was washed two times with PBS, filtered with 40 μm strainer, suspended with CellBanker 1, and gradually freezed at -80 ºC by using CoolCell (Thermo Fisher Scientific) for long term storage. Stored spleen cells were thawed and washed just before use for processing by BD Rhapsody. 2000 mouse spleen 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, and the remained half of the beads were subjected to library generation by BD official WTA workflow. 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 SP and a Novaseq 6000 sequencer (Illumina) according to the manufacture's instructions. 13% PhiX spike-in was used for sequencing.