title: GSM7162844: HC_B_cells_ADT; Homo sapiens; RNA-Seq
library_construction_protocol: B cells from fresh PBMCs were isolated using the Miltenyi No Touch B cell Enrichment Kit.   Cells   were   then   divided   in   half,   one   for   scRNA-sequencing   and   one   for   flow verification/comparison.   Samples   for   scRNA-seq   were   stained   with   Total-Seq   antibodies (Biolegend) at 1ug/ 106 cells for the following surface markers: lgD, lgM, CD27, CXCR5, and CD21 for 20 minutes on ice, washed twice with FACS buffer, and then resuspended in 1X PBS. Cells were then prepared for scRNA-seq using the 10X Genomics platform per manufacturer's instructions  (see  below).  For  the  flow  cytometry  parallel  samples,  B  cells  were  stained  with antibodies  of  the  same  clone  as  Total-Seq  antibodies  using  the  following  panel:  anti-CD27-BUV563,  anti-CD19-BV510,  anti-lgM-BV650,  anti-lgD-FlTC,  anti-CXCR5-PE/Dazzle594,  and anti-CD21-PE/Cy7 (all Biolegend) for 15 minutes on ice, washed twice with FACS buffer, and then fixed in 2% PFA in preparation for flow cytometry. A single cell suspension of B cells previously stained with Total-Seq antibodies was prepared at 1000 cells/ul for a capture of 10,000 cells on the Chromium Next GEM Chip G for 5' capture gel beads. Following single cell capture and GEM-RT Cleanup, as described in the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 User Guide Rev F, a cDNA amplification reaction was prepared  with  the  addition  of  an  appropriate  primer  to  amplify  the  Total-Seq  antibody  oligo (feature  barcode)  sequences  in  addition  to  captured  mRNA.  After  cDNA  amplification,  the supernatant  from  the  0.6X  SPRIselect  size  selection  was  saved  and  used  downstream  for analysis of the feature barcodes contained in that smaller size fraction. The 5' Gene Expression (GEX) Library Construction was done as described in the previously mentioned User Guide for the  mRNA  GEX  Library.  For  the  Feature  Barcode  (ADT)  Library  preparation,  the  CITE-seq protocol from the New York Genome Center Technology Innovation  Lab Version: 2019-02-13 was  followed  using  the  retained  supernatant  from  the  cDNA  cleanup.  Unique  indexes  were added to the 3' and 5' ends of the ADT library molecules with 2X PCR NEB High Fidelity with Q5 Polymerase Master Mix for downstream demultiplexing and data analysis.