GSM6784660: LEW1 - TLH - scRNAseq; Rattus norvegicus; RNA-Seq
Samples were prepared as outlined by the 10x Genomics Single Cell 3′ v2 Reagent Kit user guide. Briefly, the samples were washed twice in PBS (Life Technologies) + 0.04% BSA (Sigma) and re-suspended in PBS + 0.04% BSA. Sample viability was assessed via Trypan Blue (Thermo Fisher) and using a haemocytometer (Thermo Fisher). Following counting, the appropriate volume for each sample was calculated for a target capture of 10,000 cells for TLH and 9000 cells for immune-enriched samples. Samples below the required cell concentration as defined by the user guide (i.e., <400 cells/µl) were pelleted and re-suspended in a reduced volume and counted again using a haemocytometer prior to loading onto the 10x Genomics single-cell-A chip. After droplet generation, samples were transferred onto a pre-chilled 96-well plate (Eppendorf), heat-sealed and reverse transcription was performed using a Veriti 96-well thermal cycler (Thermo Fisher). After the reverse transcription, cDNA was recovered using Recovery Agent provided by 10x followed by a Silane DynaBead clean-up (Thermo Fisher) as outlined in the user guide. Purified cDNA was amplified for 12 cycles before being cleaned up using SPRIselect beads (Beckman). Samples were diluted 4:1 (elution buffer (Qiagen):cDNA) and run on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. cDNA libraries were prepared as outlined by the Single Cell 3′ Reagent Kits v2 user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration (as recommended by 10X Genomics).  The molarity of each library was calculated based on library size as measured using a bioanalyzer (Agilent Technologies) and qPCR amplification data (Kappa/Roche).  Samples were pooled and normalized to 10 nM, then diluted to 2 nM using elution buffer (Qiagen) with 0.1% Tween20 (Sigma). Each 2 nM pool was denatured using 0.1 N NaOH at equal volumes for 5 min at room temperature. Library pools were further diluted to 20 pM using HT-1 (Illumina) before being diluted to a final loading concentration of 14 pM. 150 μl from the 14 pM pool was loaded into each well of an 8-well strip tube and loaded onto a cBot (Illumina) for cluster generation. Samples were sequenced on a HiSeq 2500 with the following run parameters: Read 1—26 cycles, read 2—98 cycles, index 1—8 cycles. A median sequencing depth of 50,000 reads/cell was targeted for each sample.