title: GSM6738023: Hashtagged_scCITEseq_sample_2; Homo sapiens; OTHER
library_construction_protocol: Fresh human biospecimens were initially digested into a single cell suspension and cryopreserved in Bambanker Freezing Medium. On the day of the 10X Genomics protocol, cryopreserved samples were thawed, sorted for DAPI- CD45+ CD3+ T cells, hashtagged, stained with TotalSeq C v.1.0 cocktail from Biolegend. Library was prepared using Chromium Single Cell 5ʹ v2 protocol (10x Genomics). Briefly, cDNA amplification included 14-16 cycles and 5.8 ng-20 ng of the material was used to prepare sequencing libraries with 16 cycles of PCR. Amplification products generated using the methods described above included both cDNA and feature barcodes tagged with cell barcodes and unique molecular identifiers. Smaller feature barcode fragments were separated from longer amplified cDNA using a 0.6X cleanup with aMPure XP beads. Libraries were constructed using the Chromium Single Cell 5' Feature Barcode Library Kit according to the manufacturer's protocol with 9 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE26/91 or PE28/91 run using the NovaSeq 6000 SP or S2 Reagent Kit. An average of 60 million paired reads was generated per sample.