title: GSM9076308: SG05, MULTI-seq, LMO capture 2; Homo sapiens; OTHER
library_name: GSM9076308
library_construction_protocol: Cells previously isolated from bone marrow and spleen of mice treated transplanted with SPLINTR barcoded Molm-13 cells and treated with either Vehicle or Combination and T0 (pre-transplantation population) were thawed at 37 C in a water bath before being resuspended in FACs buffer (0.5% FBS in PBS). Viable, SPLINTR barcoded cells were sorted using propidium iodide and GFP. Samples were hashtagged with MULTI-seq lipid-modified oligos (LMOs) following manufacturers instructions. LMO-tagged cells were pooled before measuring viability using trypan blue. Following this, cells were captured using 10X Genomics 3' GEX workflow following manufacturers instructions. 10X Genomics 3' GEX and MULTI-seq LMO libraries were contstructed following their respective manufacturer's instructions. For the SPLINTR library, full length cDNA from the 10X GEX library was used as input. Barcode sequences were amplified using primers which bind to the P5 adaptor and TruSeq sequences read 1 (forward primer) and constant region of barcode, i7 index, TruSeq read 2 and P7 adapter. Libraries were cleaned using 0.7X SPRI beads before runing on an agarose gel to ensure amplicon size ~450bp and quantifiying via Quibit DNA Broad Range and Agilent Tapestation DNA High Sensitivity. The 10X 3' GEX library sequencing was performed on the Illumina NovaSeq6000 with 100bp paired-end chemistry targeting 70 million reads per cell. The MULTI-seq library sequencing was performed on the Illumina NextSeq6000 with 100bp single-end chemistry targeting 70 million reads per sample. The SPLINTR barcode library sequencing was performed on the Illumina NextSeq2000 with 100bp paired-end chemistry targeting 5 million reads per cluster.