title: GSM8942316: Nuclei from P7 pooled from mouse kidneys; Mus musculus; RNA-Seq
library_name: GSM8942316
library_construction_protocol: The kidney pairs of two male and two female mice at each age (P0, P2, P7, P14, P21, 6wk) were dissected in cold PBS. Cortical regions were partially removed to enrich for medullary cell types. Kidneys were minced into small cubes, dounced 5 times with the loose pestle in Nuclei Lysis Buffer 1 (NLB1) (4 ml of nuclei EZ lysis buffer, 20 µl of RNasin Plus, 20 µl of SUPERaseIN, 1% BSA, cOmplete ULTRA tablet), and passed through a 200 µm strainer. The sample was then dounced 10 times with the tight pestle, incubated for 1 minutes in NLB1 on ice, passed through a 40 µm strainer, and centrifuged at 500g for 1 minutes at 4˚C. The pellet was resuspended in NLB2 (4 ml of Nuclei EZ Lysis Buffer, 4 µl of RNasin Plus, 4 µl of SUPERaseIN, 1% BSA, cOmplete ULTRA tablet), incubated on ice for 5 minutes, and centrifuged at 500g for 5 minutes at 4˚C. The pellet was resuspended in nuceli suspension buffer (2 ml of calcium/magnesium free PBS, 2 µl of RNasin Plus, 2% BSA) and passed through a 5 µm strainer. The nuclei were counted by hemocytometer and diluted to 1200 nuclei/uL. The resulting nuclei suspensions were submitted to UTSW Next Gen Sequencing Core for library preparation and sequencing using the Chromium Single Cell 3' Prime Gene Expression kit (10x Genomics) on an Illumina NextSeq 2000.