GSM8900401: Purkinje cells P7 control biol rep6; Mus musculus; RNA-Seq
Sagittal slices of vermal cerebellar tissue were transferred into a recording chamber and maintained at 34C under continuous perfusion with equilibrated aCSF. For P7 Pcp2cre/+;Kir2.1 mice, Purkinje cells were selected based on their expression of Kir2.1-mCherry. Neurons were extracted into a glass pipette using negative pressure and 50 cells were sampled across the cerebellar cortex. For each genotype, eight biological samples were used for RNA sequencing. Cells were collected in a mild hypotonic lysis buffer composed of 0.2% Triton-X and 2U/µl of RNAse inhibitor. Isolated cells were kept on ice during the collection process or immediately stored at -80°C for RNA extraction. For each biological sample, cDNA libraries were generated using the Smart-seq2 method. In brief, cells were lysed, and polyA+ RNA was reverse-transcribed using an oligo(dT) primer. Template switching by reverse transcriptase was achieved by using an LNA containing TSO oligo. The reverse transcribed cDNA was pre-amplified with primers for 18 cycles, followed by clean-up. Tagmentation was performed on 1 ng of the pre-amplified cDNA using Illumina Nextera DNA Flex kit. The tagmented library was extended with Illumina adaptor sequences by PCR for 12 cycles and purified. The resulting sequencing library was measured on Bioanalyzer and equimolar loaded onto a flowcell and sequenced according to the Illumina TruSeq v3 protocol on the Illumina HiSeq2500 platform with a single read 50 base-pairs and dual 9 base-pairs indices.
GEO
GSM8900401_r1