GSM7680519: P0 Kidney LPD replicate 1; Mus musculus; RNA-Seq
For the E14.5 samples - two groups of kidneys five E14.5 kidneys, from 2 different females, per diet were pooled together and processed to obtain single-cell suspension. Approximately 10k single cells (per sample) were emulsified with Single Cell 3' V3 beads (10X Genomics) and libraries generated on a 10X Chromium instrument. For the P0 samples - one snap frozen male and one snap frozen female kidney were placed in a cold Kimble dounce homogeniser and gentle nuclear extraction was performed using a nuclear lysis buffer (Sigma) dosed with RNAse inhibitors while grinding the kidneys. After homogenisation, nuclei were pelleted at 300 x g for 3 minutes, supernatant aspirated and resuspended in phosphate buffered saline containing RNAse inhibitors, centrifuged once more, aspirated, resuspended in PBS containing RNAseq inhibitor. Nuclei were stained with DAPI and flow sorted to enrich nuclei and remove any remaining particulates. Nuclei were finally processed by emulsion with 10X Genomics 3' V3 beads, and library generation on a 10X chromium instrument. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, kidney cells or nuclei were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.