title: GSM6050543: PBMCs, COVID-19, WTA, Ab-Seq, SMK [B7]; Homo sapiens; RNA-Seq
library_construction_protocol: PBMCs were isolated from the healthy (n=4), COVID-19 positive (n=16) and recovered (n=13) individuals using BD vaccutainers Cell Preparation Tube (CPT). Samples were stained with BD Sample Multiplexing kit for Human, wherein unique oligo sequences were attached with a universal antibody. Further, the samples were pooled and stained using a pool of BD Ab-Seq oligo attached antibodies. After counting the tagged cells, an average of 30000 cells for each batch were loaded on to the BD Rhapsody cartridge to capture the single cells using BD Rhapsody Express Single-Cell Analysis System. Each Sample Tag has common 5' and 3' ends and the Sample Tag sequence: GTTGTCAAGATGCTACCGTTCAGAG[Sample Tag sequence]AAAAAAAAAAAAAAAAAAAAAAAAA. The Sample Tag oligo sequences and the Ab-Seq oligo sequences are available as supplementary files. The sample id, the experimental group, batch, corresponding sample tags and barcodes are available as supplementary data. Library was prepared using BD Rhapsody WTA amplification kit. Briefly, cDNAs were prepared on the cell capture beads obtained from the BD Rhapsody cartridge. The Ab-Seq and sample tag oligos were separated from the Exonuclease I treated beads by denaturation, while the cDNA of the poly-A transcripts remained on the beads. A random priming and extension was performed on the beads, followed by Ampure XP based cleanup, amplification, secondary Ampure XP cleanup, indexing and a final dual-sided Ampure XP cleanup to make the WTA library. On the other side, the Ab-Seq/sample tag oligos were amplified using universal oligos followed by a Ampure XP cleanup. The Ab-Seq library prepared after an index-PCR, followed by a Ampure XP cleanup, while the sample tag oligos were enriched by an aditional PCR, followed by a Ampure XP cleanup. Finally, the SMK library was constructed using Index PCR of the enriched sample tag oligos, followed by final Ampure XP cleanup. The barcodes sequences and barcodes used for wach batch are available in supplementary file. Following the final bead purification, all three libraries (per batch) were pooled in order to obtain a 30000 reads/cell for WTA library, 20000 reads/cell for Ab-Seq library and 120 reads/cell/sample tag/batch for the SMK library. Libraries of multiple batches were pooled based on the index sequences used during Index PCR steps.