GSM8170128: B cells, GEX; Mus musculus; RNA-Seq
Spleen of unimmunized mice (KO and Ctrl) and day-4 SRBC-immunized mice (KO and Ctrl) were recovered. Splenic B and CD4+ T cells were magnetically isolated with Mouse B cell isolation kit and naive CD4+ T cells respectively (Miltenyi). Library was performed according to the manufacter's instructions (single cell 5' V2 protocol, 10X genomics with feature barcode technology). Briefly, B and CD4+ T cells were resuspended in the master mix and loaded together with partioning oil and gel beads into the chip to generate the gel bead-in emulsion (GEM). The poly-A RNA from the lysate containing in every single GEM was retrotranscripted to cDNA, which contains an illumina R1 primer sequance, unique molecular identifier (UMI) and the 10X barcode. The pooled bacoded cDNA was then cleaned up with Silane Dynabeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. In parallele, we performed cell surface protein/immune receptor mapping library (HTO) to multiplex B cells from control (Cre) and KO mice unimmunized (D0) or after 4 days (D4) SRBC immunization in the same library, and to multiplex T cells from control (Cre) and KO mice unimmunized (D0) or after 4 days (D4) SRBC immunization in a second library.