GSM8162165: RNA-seq after pulldown of P180GFPbio in primary cortical neurons; Rattus norvegicus; RNA-Seq
For RNA-immunoprecipitation, the same procedure was followed with a few adaptations. SUPERase-in RNase Inhibitor (Thermo Fisher) was added to lysis and wash buffers. After bead incubation and 4 washes, RLT buffer (QIAGEN) + β-mercaptoethanol was added to the beads and incubated with vortexing for 2 minutes. The sample was then separated from the beads on a magnetic rack and transferred to a RNeasy column for RNA isolation (QIAGEN) according to protocol. RNA was stored at -80°C until processing for RNA-sequencing. Isolated RNA as described above was used for library preparation and sequencing. mRNA was processed as described previously, following an adapted version of the single-cell mRNA seq protocol CEL-Seq2.75,76 In brief, samples were barcoded with CEL-seq primers during a reverse transcription and pooled after second strand synthesis. The resulting cDNA was amplified with an overnight in vitro transcription reaction. From this amplified RNA, sequencing libraries were prepared with Illumina Truseq small RNA primers. The DNA library was paired-end sequenced on an Illumina Nextseq 500, high output, with a 1x75bp Illumina kit (R1: 26 cycles, index read: 6 cycles, R2: 60 cycles).