GSM6069080: Single-cell RNA-Sequencing b-cells expressing CEBPa AroPerfect IS10; Homo sapiens; RNA-Seq
GSM6069080
To induce C/EBPα mediated B-cell to macrophage transdifferentiation, infected RCH-rtTA cells were seeded at 0.3x106 cells/ml in RCH culture medium supplemented with 10 ng/ml of each, IL-2 (200-03, Preprotech) and CSF-1 (315-03B, Preprotech), as well as 2 µg/ml of Doxycycline. The macrophage transdifferentiation was monitored by flow cytometry. One week after induction of C/EBPα mediated B-cell to macrophage transdifferentiation, cells were collected and washed twice in PBS to remove dead cells and debris. Cells were resuspended in solution at a density of 700 cells/µl. We used a Chromium Next GEM Single Cell 3' technology for generating gene expression libraries from single cells. Briefly, gel beads-in-emulsion (GEMs) are generated by combination of barcoded Single Cell 3' v3.1 Gel Beads, a Master Mix containing cells, and Partitioning Oil on a Chromium Next GEM Chip G. To achieve single cell resolution, cells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contain no cell, while the remainder largely contain a single cell. Immediately following GEM generation, gel beads were dissolved, primers were released, and any co-partitioned cell was lysed. Primer (containing an Illumina TruSeq Read 1, 16 nt 10x Barcode, 12 nucleotide unique molecular identifier and 30 nucleotide poly-dT sequence) were mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produced barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken and pooled fractions were recovered. Silane magnetic beads were used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA was amplified via PCR to generate sufficient mass for library construction. cDNA was analyzed using Agilent Bioanalyzer assay (ref. 5067-4626, Agilent) to check size distribution profile and quantification. Only 25% of cDNA was used to 3' Gene Expression Library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size. TruSeq Read 1 (read 1 primer sequence) was added to the molecules during GEM incubation. P5, P7, a sample index, and TruSeq Read 2 (read 2 primer sequence) were added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. Final libraries were analyzed using Agilent Bioanalyzer assay to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification K