Name: GSM7709783
Instrument: Illumina NovaSeq 6000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Splenic and bone marrow cells (tibia, femur and iliac crest) were isolated from C57BL/6J mice immunized with NP-KLH, alum and LPS on 14, 21 and 35 d.p.i.. Splenocytes and BM cells were washed and prepared as single-cell suspensions in MACS buffer. Enriched CD138+ cells were obtained by using Easysepä Release Mouse Biotin Positive Selection Kit. Cell concentration was adjusted to 100 x 106 cells per mL and incubated with 50 μL of normal rat serum for 5 minutes. Biotinylated anti-CD138 (281.2) antibody (1.0 μg/mL) was added to the cells and incubated for 5 minutes. Next the cells were incubated (5 minutes) with Easysepä Biotin positive selection antibody (50 μL/mL) followed by addition of Easysepä Rapidspheres (100 μL/mL) (5 minutes). MACS buffer was added to the cocktail and an Easysepä magnet was used to remove cells that were not bound to rapidspheres. After three washes, CD138+ cells were obtained by incubating with Easysepä Release Buffer for 5 minutes and placing the cells in a magnet to release them from the Rapidspheres. Purity of enriched CD138+ cells was determined by flow cytometry after labeling a small fraction of the cells with viability dye eFluor 780 (eF780), APC Streptavidin and PE-Dazzle Red 594 B220 (RA3-6B2) for 30 minutes at 4˚C. For the isolation of CD138+ subsets by flow cytometry, enriched CD138+ cells after Rapidspheres release, were labeled with viability dye eFluor 780 (eF780), APC-Streptavidin, PE-Dazzle Red 594 B220 (RA3-6B2), PerCP-Cy5.5 CD44 (IM7) and FITC CD11a (M17/4) for 30 minutes at 4˚C and were sorted as B220+CD138+CD44-CD11a-, B220+CD138+CD44+CD11a+, B220intCD138+CD44+CD11a+ and B220-CD138+CD44+CD11a+ subsets using FACSAria II (BD) with 70 μm nozzle at 4˚C. Enriched CD138+ splenocytes or flow sorted CD138+ splenic and BM subsets were suspended in 5% FBS and loaded along with oil and beads containing unique barcodes onto the Chromium controller (10x Genomics) to form an emulsion. Single cells contained in oil droplets were then lysed and mRNA was captured by the beads and subjected to reverse transcription and cDNA amplification to generate libraries for sequencing, according to the manufacturer's protocol, as described previously2. From the cDNA libraries, one aliquot was used for fragmentation and preparation for transcriptome profiling, and another for amplification of VDJ regions using the BCR kit. Samples were then sequenced using a NovaSeq 6000 (Illumina) in which 30,000 and 5,000 reads were generated with the mRNA and BCR libraries respectively.