GSM5453390: macrophages; Homo sapiens; RNA-Seq
scRNA-seq was performed in the Dresden-concept Genome Center, Center for Regenerative Therapies Dresden (CRTD), Cluster of Exellence at TU Dresden, Germany. Cells were mixed with the reverse transcription mix before loading them in a Chromium Single Cell G Chip on the 10X Genomics Chromium device and processed further following the guidelines of the 10x Genomics user manual (v3.1). The droplets were directly subjected to reverse transcription, the emulsion was broken and cDNA was purified using silane beads. After amplification of cDNA with 11 cycles using primers to enrich cDNA as well as Totalseq-B hashtag, it underwent a cleanup, including a fractionation of small fragments (up to 400 bp) to enrich the hashtag sequences and larger fragments (>400 bp) to separate cDNA fragments. RNA libraries were prepared for sequencing using standard 10x protocols. For the gene expression library 10 µl cDNA were used involving fragmentation, dA-Tailing, adapter ligation and a 12 cycles indexing PCR was performed based on the manufacturer's protocol. The hashtag library was prepared with 5 µl FBC-fraction by a 12-cycles index PCR. For both libraries Chromium SC NextGem 3 Lib prep v3.1 was used.