GSM8172655: FLM3_rna; Homo sapiens; RNA-Seq
First, the cell suspensions were fixed in 1ml of fixation buffer for 16-24 hours at 4ºC according to the 10X genomics fixation protocol and later resuspended in quenching buffer. Next, following the 10X Genomics Chromium Fixed RNA profiling protocol, the fixed cells were hybridized with individual probes and later on washed and counted on the Countess II Automated Cell Counter. If multiplexing, the samples were pooled based on the limiting sample (the one with lowest number of cells). As a consequence, in the pool we had the same cell counts per sample. After several washes of the pool, we measured the pool stock concentration. Based on the manufacter's recommendations and our cell stock concentration, the targeted cell recovery was set. Afterwards, the GEM generation took place in the Chromium Next GEM Chip Q. In addition, the GEMs were incubated, pre-amplified and cleaned with the SPRIselect beads, as advised by the manufacter before the library construction. The library construction was made as described in the 10X Chromium Fixed RNA Profiling protocol. For that, after the pre-amplification PCR clean-up, we proceeded with the Sample Index PCR, in which individual indexes from the Dual Index TS Set A were used for each of the samples. The number of cycles in the PCR was determined based on the targeted cell recovery. After the PCR, the samples went through a size selection clean-up with the SPRIselect beads and eluted in the Qiagen Buffer EB. The Agilent TapeStation High Sensitivity was run to check the traces of the libraries and the Qubit fluorometer was used to quantify their concentrations. Lastly, the libraries were sequenced on the Illumina NovaSeq Platform.