Determination of astrocyte transcripts regulated by neuronal contact

We co-cultured P0 TdTomato-expressing hippocampal neurons with wild type cortical astrocytes for 2, 4 or 7 days in vitro and then separated the populations using FACS. We then compared transcripts from co-cultured with those from astrocytes cultured alone Overall design: Astrocytes were grown in culture until confluent on 15cm plates and then switched to N2 media. 1x10^6 P0 hippocampal neurons were plated on experimental plates and grown for indicated time (2, 4 or 7 days in vitro). At the indicated time, cells were trypsanized and underwent FACS sorting using the TdTomato emission peak of 581nm. Control astrocytes were used to set TdTomato- gates. Total RNA was extracted with Trizol and rRNA was removed using the Ribogone kit. cDNA synthesis and indexed Illumina libraries were made using the SMARTer Universal Low Input RNA kit for Sequencing and the SMARTer Low Input Library Prep Kit and sequenced on HiSeq 2000