GSM5035596: WT NP+IgE+ Bcell#6; Mus musculus; RNA-Seq
We transferred IL-13 sufficient or deficient OT-2 TH2 into IgE reporter (Verigem) mice followed by immunization with NP-OVA. At 7 day post immunization, NP specific IgE producing B cells were sorted by flow cytometer. For scRNA-seq analysis, the single-cells were sorted into each well of 96 well plates using FACS Aria III (BD). The single cells are lysed in a buffer that contains dNTPs and oligo(dT)-tailed oligonucleotides with a universal 5'-anchor sequence. RT is performed, which adds 2–5 untemplated nucleotides to the cDNA 3' end. A template-switching oligo (TSO) is added, carrying 2 riboguanosines and a modified guanosine to produce a LNA as the last base at the 3' end. After the first-strand reaction, the cDNA is amplified using a 25 of cycles. Next, Tn5 tagmentation is used to construct sequencing libraries from the amplified cDNA (Nextera XT DNA Library Preparation Kit, illumina). The sequencing was carried out using NovaSeq6000 equipment (SP type, 150PE, Illumina).
GEO Accession
GSM5035596