scRNA-seq using the 10x Genomics platform

scRNA-seq using the 10x Genomics platform

Version of kit used: Chromium Single Cell 3’ kit (v2)

Growth and sampling

-    Pre-grow cells in 3 mL YPMAL10% in dilute conditions for 1 overnight at 30°C in a spinning wheel, making sure final cell counts remain under 2*10⁶ cells/mL

-    Refresh media and make serial dilution so that after the 2^nd overnight (30°C, spinning wheel), cultures are at a cell count of ~2*10⁶ cells/mL

-     Wash and inoculate cell cultures into 50 mL YPGLU5% to reach a starting cell density of 10⁴ cells/mL and grow for 12 hr (shaking incubator, 30°C – 200 rpm)

-     Wash to YPMAL10% and monitor cell counts continuously throughout lag phase

-    For each sample taken during the lag phase, immediately freeze 0.5 mL of culture mixed with 0.5 mL of ice-cold glycerol50% at -80°C until the running day of the Chromium device

Running 10x Genomics Chromium device

-    Prepare 100x zymolyase stock solution (100mg/mL) in the buffer of the QuantiTect reverse transcriptase; and filter sterilize (syringe filter - 0.2 µm pores). Keep on ice and avoid freeze-thaw cycle

-    Thaw cell cultures on ice, measure cell count and pellet the cells (3000 rpm, 3’). Resuspend into ice-cold filter-sterilized PBS to reach the desired cell count, according to the 10x Genomics Chromium Single Cell 3’ protocol. Limit time between thawing and the Chromium run as much as possible

-    From here on, follow the protocol of the 10X Genomics Chromium device; except add the zymolyase solution to the reverse transcription master mix (replacing 1 µl of water in the master mix with 1 µl of 100x zymolyase stock solution)

Buffers & growth media used

-    YPMAL10%:
Autoclave YP2x solution (20 g/L yeast extract, 40 g/L bacteriological peptone) and filter-sterilize MAL20% (210.5 g/L maltose monohydrate). Mix ½ of YP2x with ½ of MAL20% to reach desired concentrations
-    YPGLU5%:
Autoclave YP2x solution (20 g/L yeast extract, 40 g/L bacteriological peptone) and filter-sterilize GLU20% (220 g/L glucose monohydrate). Mix ½ of YP2x with ¼ of autoclaved dH₂O and ¼ of GLU20% to reach desired concentrations
-    PBS:
Phosphate buffered saline, autoclaved, pH 7.2

Sequencing and initial analysis

The prepared libraries were sequenced on one run of Illumina NextSeq and two lanes of Illumina HiSeq 3000.

-    Convert BCL files from Illumina sequencer to fastq files using the 10x Genomics’ Cell Ranger pipeline with mkfastq option

-    Map, filter reads and carry out UMI counting using Cell Ranger with count option; fastq files per sample are arguments for the count command, and set the expected number of cells option to the expected value based on the calculation table in the protocol of the 10X Genomics Chromium device

-    If samples need to be aggregated, use the aggr option of Cell Ranger without any aggregation normalization of total UMI’s per cell (we did not use normalization of total UMI’s since we know that RNA content of cells drops during lag phase)

-    Filter out cells with excess mitochondrial reads and possible doublets (decide on thresholds using the VlnPlot function of the Seurat package to inspect the violin plots of distribution values)

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