Journal: bioRxiv

Article Title: HIV-SEQ Reveals Global Host Gene Expression Differences Between HIV-Transcribing Cells from Viremic and Suppressed People with HIV

doi: 10.1101/2024.12.17.629023

Figure Lengend Snippet: A. Schematic illustrating the genomic location of HIV-specific capture sequences of HIV-seq. The name and nucleotide position (based off HXB2 annotation) of each of capture sequence are labeled in green. B. Schematic of HIV-seq protocol. PBMCs from PWH are enriched for CD4+ T cells and then labeled with CITE-seq antibodies to enable subsequent surface phenotyping. After cell encapsulation using a 10X Chromium instrument, custom-designed HIV-specific capture sequences described in panel A that have been appended to a non-poly(dT) PCR handle are spiked in with the poly(dT) oligos and incorporated into the 10X Genomics’ Chromium Next GEM Single Cell 5’ workflow. Each ‘Single Cell 5’ Gel Bead’ features an Illumina R1 sequence (‘read 1’ sequencing primer), a 16 nucleotide (nt) 10X Barcode (BC), a 10 nt unique molecular identifier (UMI), and a 13 nt template switch oligo (TSO). Reverse transcription is primed off both poly(dT) as well as the HIV capture sequences (i). Following template switching and transcript extension (ii, iii), barcoded cDNA libraries corresponding to both gene expression (GEX) and antibody-derived tags (ADT, for CITE-seq) are processed through the standard 10X workflow, and then sequenced. Data analysis was performed using the Seurat pipeline, SeqGeq software, and custom scripts.

Article Snippet: The standard 10X Genomics’ 5’ scRNA-seq workflow entails droplet encapsulation of individual cells, followed by capture and reverse transcription of polyadenylated transcripts using poly(dT) oligos.

Techniques: Sequencing, Labeling, Encapsulation, Reverse Transcription, Expressing, Derivative Assay, Software