GSM8178802: 392-WT-CD; Mus musculus; RNA-Seq
Animals were perfused with ice-cold PBS prior to brain dissection and snap frozen. Nuclei from P40 female hippocampi with acceptable RNA quality (RNA Integrity Number (RIN) > 7 with Agilent RNA 6000 Nano kit (Agilent, 50671511)) were isolated according to Frankenstein protocol for nuclei isolation from fresh and frozen tissue. Nuclei were sorted via fluorescence-activated nuclei sorting (FANS) by a DAPI+ gate and two further gates of NeuN positive and NeuN negative nuclei (anti-NeuN antibody from Abcam, ab190195), in order to obtain 5000 neurons and 5000 glia per sample, respectively. The nuclei were processed with Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics, 1000268) generate the cDNA libraries for this study. Gel Bead-in-Emulsions (GEMs) were generated with chip G (10x Genomics, 1000120) on the microfluidic platform 10x Genomics Chromium Single Cell Controller. Next, the samples were uniquely barcoded to distinguish the mRNAs (each having unique molecular identifier (UMI)) from different nuclei. The cDNA was extracted with Dynabeads MyOne SILANE beads (Invitrogen, 370-12D) and amplified by PCR. The PCR product was purified with SPRIselect reagent (Beckman Coulter, Cat #B23318). Subsequently, the 3' gene expression library was constructed with primers for Illumina NGS platform and Dual Index Kit TT set A (10x Genomics, 1000215) which allows individual sample identification.