title: GSM4158190: L27389: Cortex_gfp_3; Mus musculus; RNA-Seq
library_construction_protocol: Cells were sorted directly in RNA lysis buffer. The RNA-seq library preparation was performed on the basis of Picelli et al. 2013  with some modifications. Into a PCR tube containing 2 µl of a 0.2% Triton-X 100 solution in nuclease free water and 4 U murine RNase Inhibitor (NEB), 180-300 cells were sorted using a BD FACS Aria III. The cells were frozen at -80°C to enhance the lysis effect. After thawing, the RNA in the cell lysate was denatured for 3 minutes at 72 °C in the presence of 2.4 mM dNTP (Invitrogen), 240 nM dT-primer (C6-aminolinker-AAGCAGTGGTATCAACGCAGAGTCGACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN, where N represents a random base and V any base beside thymidine) and 4 U RNase Inhibitor (NEB). The reverse transcription and addition of the template switch oligo was performed at 42°C for 90 min after filling up to 10 µl with RT buffer mix for a final concentration of 1x superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgCl2, 1 µM TSO-primer (AAGCAGTGGTATCAACGCAGAGTACATrGrGrG, where rG stands for ribo-guanosine), 9 U RNase Inhibitor and 90 U Superscript II. The reverse transcriptase was inactivated at 70 °C for 15 min. The single stranded cDNA was subsequently amplified using Kapa HiFi HotStart Readymix (Roche) at a 1x concentration together with 250 nM UP-primer (AAGCAGTGGTATCAACGCAGAGT) under following cycling conditions: initial denaturation at 98°C for 3 min, 17 cycles [98°C 20 sec, 67°C 15 sec, 72°C 6 min] and final elongation at 72°C for 5 min. The amplified cDNA was purified using 1x volume of Sera-Mag SpeedBeads (GE Healthcare) resuspended in a buffer consisting of 10 mM Tris, 20 mM EDTA, 18.5 % (w/v) PEG 8000 and 2 M sodium chloride solution. The cDNA quality and concentration was determined with a Fragment Analyzer (Agilent). For library preparation 1 ng cDNA was tagmented using 0.5 µl Tagment DNA Enzyme and 1x Tagment DNA Buffer (Nextera DNA Library Prep Kit, Illumina), followed by an incubation step at 55 °C for 5 min. Next, Illumina indices were added during PCR (72°C 3min, 98°C 30sec, 12 cycles [98°C 10sec, 63°C 20sec, 72°C 1min], 72°C 5min) with 1x concentrated KAPA Hifi HotStart Ready Mix and 700nM dual indexing primers. After PCR, libraries were purified twice with 1x volume Sera-Mag SpeedBeads and quantified with the Fragment Analyzer.
attributes.tag: GEO Accession
attributes.value: GSM4158190