GSM8245085: 3m_Female_D28_1; Mus musculus; RNA-Seq
Exactly 600 microglia were sorted from mechanically dissociated retinas (kept ice cold) directly into lysis buffer from the SMART-seq v4 kit. All samples were collected within 2 h from cull. Each batch was randomised to include a mix of all conditions to mitigate batch effects of sort and cDNA preparation. Samples were collected over two days, with all samples collected in one day processed to cDNA preparations together (sorted samples were stored in lysis buffer on ice until all samples were collected). The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, was used for cDNA preparation and amplification (16 PCR cycles), according to the user manual, before cDNA isolation using the Agencourt AMPure XP Kit (Beckman Coulter, Brea, CA). Quantification of cDNA was performed using the Agilent High Sensitivity DNA Kit on an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA) and 150 pg of cDNA was used for library preparation using the Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA).