title: GSM2938848: RNA-seq_dopaminergic neuron sample 1; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from DA or 5-HT neurons using an RNeasy Microkit (Qiagen) following manufacturer´s instructions. RNA was treated with DNAse I (Qiagen) for 20 minutes at room temperature to prevent genomic DNA contamination. For RT-qPCR, RNA concentrations were determined by spectrophotometry (Nanodrop 2000c, THERMO Scientific). For RNA-seq, a High Sensitivity RNA ScreenTAPE analyzer (Agilent Technologies) was used to assess RNA concentrations as well as the RNA integrity number (RIN) to verify RNA quality for all tested samples. RNA was stored at−80 °C until reverse transcription or RNA-Seq. Stranded library was prepared using TotalScript RNA sequencing kit (Epicentre) following manufacturer's recommendation. For purified samples of 5HT neurons, non-stranded library were prepared using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech) following manufacturer's recommendation. 3 DA and 3 5-HT libraries were sequenced using NextSeq500 HighOutputKit v2 (300cycles) cartridge (FC-404-2004 Illumina). Sorted cells by FACS were collected in Neurobasal medium with B27 supplement (Life Technologies), 2% FBS and kept at 4°C until ATAC-Seq. Cells were centrifuged at 500 g, at 4°C during 20 min. Cells were resuspended in 25 μl of lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) during 10 min at 4°C. Then supernatant was taken out after a centrifugation at 500 g, at 4°C during 30 min. For transposase reaction, the pellet was resuspended in 25 μl of 12.5 μl 2x TN buffer; 2 μl of Tn5; 10.5 μl d'H2O and incubated at 37°C for 1 h. Then 5 μl of clean-up buffer (900 mM NaCl, 300 mM EDTA, 5% SDS) were added with 2 μl of 5% SDS and 2 μl of Proteinase K, and cells were incubated for 30 min at 40°C. Samples were then cleaned with two SPRI clean up (Agencourt © AMPure ©XP), with 68 μl of SPRI beads, eluted in 13 μl of buffer EB (Qiagen Cat No./ID: 19086). Extracted DNA concentration was measured by ScreenTAPE analyzer (Agilent Technologies). To generate libraries, PCR reactions were performed using the kapa PCR mix (Kapa biosystem) with 12.5 μl Kapa, 1 μl primers and 11.5 μl of sample, with the NextEra primers (1 μl /primer). PCR conditions were performed as described: 98°C during 2 min and then 9 cycles of 98°C during 20 s, 63°C during 30 s, 72°C during 1 min. Then, a new SPRI clean-up was made to do a size cut off of amplified PCR products and after that, DNA concentration was measured by ScreenTAPE analyzer (Agilent Technologies). Finally, a second PCR was performed with the same conditions as the first one and a last SPRI clean-up was made and library of tagged open chromatin was ready to be sequenced.
attributes.tag: GEO Accession
attributes.value: GSM2938848