title: GSM2549095: LCMV_armstrong_D05_long_single-end; Mus musculus; RNA-Seq
library_construction_protocol: Mouse LCMV: Peripheral blood was obtained from the mice at day 7 post infection (p.i.) and lymphocytes were enriched using LSM density centrifugation. Cells were prestained with a near-IR fixable live/dead marker (Life Technologies, cat# L34976) and an APC- conjugated dextramer reagent for gp33 (Immudex, cat# A2160-APC) according to manufacturer recommendations. The cells were then stained with the following antibodies: FITC 2B4 (BioLegend, cat# 133504), PerCP-Cy5.5 CD44 (BioLegend, cat# 103032), PE KLRG1 (BioLegend, cat# 138408), PE-Cy7 PD1 (BioLegend, cat# 135215), BV421 CD127 (BioLegend, cat# 135024), BV510 CD8A (BioLegend, cat# 100752). Human Donor 1: Blood samples were obtained from a donor with detectable NLV-specific CD8+ T cell response. Lymphocytes were enriched via Ficoll gradient and prestained with a near-IR fixable live/dead marker (Life Technologies, cat# L34976) and an APC-conjugated dextramer reagent (Immudex, cat# WB2132-APC). The cells were then stained with the following antibodies: FITC CD8A (BioLegend, cat# 300906), PerCP-Cy5.5 CCR7 (BioLegend, cat# 353220), PE CD3 (BioLegend, cat# 317308), BV605 CD45RA (BioLegend, cat# 304133). Human Donor 2: A whole blood sample was obtained 9 months post-vaccination and lymphocytes were enriched from whole blood via Ficoll gradient centrifugation and a CD8 negative selection magnetic bead kit (Miltenyi Biotec). Cells were prestained with a live/dead marker (Life Technologies, cat# L34976) and an APC-labeled tetramer reagent (NS4B 214–222 LLWNGPMAV, kindly provided by Dr. Rafi Ahmed). The cells were then stained with the following antibodies: FITC CD8A (BioLegend, cat# 300906), PE CXCR3 (BioLegend, cat# 353705), PE-Cy7 CCR7 (BioLegend, cat# 353226), BV421 IL2Rb (BioLegend, cat# 339009), BV510 CD3 (BioLegend, cat# 317332), BV605 CD95 (BioLegend, cat# 305627), BV780 CD45RA (BioLegend, cat# 304140). Human Donor 3: PBMC were collected 14 weeks post-boost vaccination. PBMC were thawed and prestained with a live/dead marker (Life Technologies, cat# L34976) and a PE-conjugated pentamer reagent (PE-labeled HCV NS31406–1415 (KLSALGINAV; HLA-A*0201)). The cells were then stained with the following antibodies: FITC 2B4 (BioLegend, cat# 329505), PerCP-eFluor 710 LAG3 (eBioscience, cat# 46-2239), PE-Cy7 CCR7 (BioLegend, cat# 329919), APC CD39 (BioLegend, cat# 328209), BV421 PD1 (BioLegend, cat# 329919), BV510 CD3 (BioLegend, cat# 317332), BV605 CD8A (BioLegend, cat# 301040), BV780 CD45RA (BioLegend, cat# 304140). Single cell lysates were converted to cDNA following capture with Agencourt RNA Clean beads using the SmartSeq2 protocol. The cDNA was amplified using 22-24 PCR enrichment cycles prior to quantification and dual-index barcoding with the Illumina Nextera XT kit. The libraries were enriched with 12 cycles of PCR, then combined in equal volumes prior to final bead cleanup and sequencing.
attributes.tag: GEO Accession
attributes.value: GSM2549095