GSM5930760: control axon_Rep5; Mus musculus; RNA-Seq
NucleoSpin RNA PLUS XS Kit was used for RNA extraction. Samples from axonal compartment and somatodendritic compartment were collected in lysis solution and incubated at 4° (protocol adapted from Bio-protocol 9(14): e3312, DOI:10.21769/BioProtoc.3312). Then, lysed samples were treated to digest the DNA and the RNA that was eluted in RNAse-free water. Quant-iT RiboGreen RNA Kit was used for the quantification of total RNA. RQN was >=9.7 for all samples. The NEBNext Low Input RNA Library Prep Kit for Illumina was used to process the sample(s). The sample preparation was performed according to the protocol "NEBNext Low Inputr RNA Library Prep Kit for Illumina" (NEB #E6420S/L). Briefly, cDNA was syntesized and amplified from poly A tailed mRNA. This was used for ligation with the sequencing adapters and PCR amplicification of the resulting product. The quality and yield after sample preparation was measured with the Fragment Analyzer (see appendices). The size of the resulting products was consistent with the expected size distribution (a peak between 200-400 bp). Low input sample preparations including unique dual indexing and unique molecular identifiers (UMIs). RNA sequencing (polyA enriched) of 24 totalRNA samples (mouse), using Illumina NovaSeq6000 sequencing, Paired-End, 150 bp. Per sample ~9 Gb, 30 million Paired-End reads.