GSM8574962: in vivo Ago2 WT brain cells, replicate 2; Mus musculus; RNA-Seq
Snap frozen hemispheres of mice at age P14 were minced on ice into 1 mm x 1 mm pieces, dounced in 2 ml ice cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2 U/ul recombinant RNase inhibitor (Takara)) and lysed on ice for 5 minutes. Lysis was stopped by adding 10 ml of ice-cold PBS-BSA (1% BSA, 0.2 U/ul RNase inhibitor), the suspension was filtered through a 40 mm cell strainer and centrifuged at 4°C, 500g for 5 minutes. Nuclei were washed two times with PBS-BSA, counted and assessed for quality. Immediately before loading nuclei on the 10x Chromium, nuclei were washed and concentrated to remove potential ambient RNA. Post wash between 2000 and 5000 nuclei were loaded onto the 10X Chromium System for nuclei encapsulation as per the manufacturer's instructions in Chromium Single Cell 3' Reagents kit v3 user guide RevC. Libraries were prepared using Single Cell 3' v3 chemistry. Briefly, RNA was reverse transcribed in the gel bead emulsion and cDNA amplified adding three additional PCR cycles to compensate for low RNA content of nuclei as compared to cells. Subsequently, libraries were fragmented and indexed. Final libraries were quantified and assessed for fragments size using the Agilent 4200 Tapestation System. Libraries were pooled and sequenced on a NovaSeq 6000 (Illumina).