title: GSM5418390: RR0511931.040820.TC#1; Homo sapiens; RNA-Seq
library_construction_protocol: Human Tregs and Tconvs from COVID-19 patients and healthy donors were magnetically isolated from frozen PBMCs using a Stem Cell kit, as CD4+CD25hiCD127lo and CD4+CD25–, respectively. Around 1,000 isolated cells were directly into lysis buffer for RNA-seq (Smart-seq2)  library preparation following the standard ImmGen low-input protocol (www.immgen.org). Samples were collected and sequenced in two cohorts. total RNA was captured and purified on RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was then selected using an anchored oligo(dT) primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3) and converted to cDNA via reverse transcription. First-strand cDNA was subjected to limited PCR amplification followed by Tn5 transposon-based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Samples were then PCR amplified for 18 cycles using barcoded primers such that each sample carried a specific combination of eight-base Illumina P5 and P7 barcodes and were pooled together prior to Smart sequencing. Smart-seq paired-end sequencing was performed on an Illumina NextSeq500 using 2 x 38bp reads with no further trimming.