GSM5322250: Astrocytes_36091 [A11]; Homo sapiens; RNA-Seq
Samples were homogenized in TRIzol and stored at -80 until processing Total RNA was isolated according to the manufacturer's instruction, quantified by Nanodrop (Thermo Fisher Scientific) and diluted to 1 ng/µl in nuclease-free water.  A total of 1 ng of RNA was added to 2.5 µl of cell lysis buffer (nuclease-free water with 0.2 % v/v Triton X-100 and 0.1 U/µl RNase inhibitor), and subjected to cDNA synthesis and amplification as described before (Cembrowski et al., 2018). Libraries were prepared using a modified Nextera XT DNA protocol (Illumina, San Diego, CA) where 5 µM P5NEXTPT5 was substituted for the i5 primers in the kit. Libraries were quantified by qPCR (Roche), normalized to 2 nM, then pooled and sequenced on a NextSeq550 flowcell with 25 bases in read 1, 8 bases in the i7 index read, and 50 bases in read 2. The control library phiX (Illumina) was spiked in at a final concentration of 15% to improve color balance in read 1.