Superscript vilo cdna synthesis kit

Quantitative PCR (qPCR) was used to examine the temporal expression of Chl1 within the developing VM. In brief, the VM was isolated from E10.5, E12.5, E14.5 and E18.5 embryos (4 litters per age) as previously described^{7 (link), 18 (link)}. The VM was additionally isolated from tyrosine hydroxylase green fluorescent protein (TH-GFP+) embryos at E12.5^{19 (link)}, to establish whether Chl1 was expressed on DA neurons (FACS isolated THGFP+ cells) or non-dopaminergic neurons (THGFP− cells) within the VM (n = 4 embryos per FACS preparation, repeated on 4 litters). For all samples RNA was isolated from using RNeasy Micro kit (Qiagen) and reverse transcribed using a SuperScript® VILO™ cDNA Synthesis Kit. qPCR performed using SYPR GreenER qPCR SuperMix Universal (Invitrogen) on a Rotor-Gene 6000 thermocycler (QIAGEN) and analyzed using the comparative ΔΔCT method^{20 (link)}. Oligonucleotide sequences were as follows:
Hprt forward, 5′-CTTTGCTGACCTGCTGGATT-3′
Hprt reverse, 5′-TATGTCCCCCGTTGACTGAT-3′
Chl1 forward, 5′-TGGAATTGCCATTATGTGGA-3′
Chl1 reverse, 5′-CACCTGCACGTATGACTGCT-3′

For genomic DNA sequencing, cells were lyzed in lysis buffer (50 mM Tris, pH = 8.0, 20 mM NaCl, 1 mM EDTA, 1% SDS) with proteinase K. DNA was extracted using phenol-chloroform isoamylalcool (ref. P2069, Sigma) and isopropanol (ref. 59309-1L, Sigma). For cDNA sequencing, RNA was extracted using the miRNA Micro Kit (QIAGEN) and reverse transcription was performed using the SuperScript ® VILO cDNA Synthesis Kit (ref. 11754–250, Invitrogen). The region encompassing the mutation was amplified by PCR using 5 or 4 sets of primers for the genomic DNA and cDNA, respectively (Supplementary Table S1) and the Kapa2G polymerase (KAPA 2G Robust HotStart PCR Kits, ref. KK5517, Clinisciences). PCR products were run on agarose gels and purified using phosphatase (FastAP, ref. EF0651, ThermoScientific) and exonuclease enzymes (Exonuclease I, EN0581, ThermoScientific). Controls were performed in the absence of reverse transcriptase. Sequencing was performed using the BigDye®TerminatorV3.1 cycle sequencing and purifications kits (ref. 4337455,4376484, ThermoFisher).

Total RNA was isolated from 10⁶ ATII cells directly after isolation or following 48 h of culture in MucilAir medium with an RNeasy MiniKit (Qiagen, Hilden, Germany). Reverse transcription was performed on 0.8 µg to 1.3 µg total RNA using the SuperScript VILO cDNA synthesis kit according to manufacturer's protocol and validated QuantiTect primer assays (Qiagen, Hilden Germany) Amplification was performed on a realplex2 mastercycler (Eppendorf, Hamburg, Germany) using the XPress Syber Green ER qRT-PCR super mix. Each reaction was carried out on cDNA from ≥three independent isolations (cDNAs were used at 1-, 10- and 100-fold dilutions). Specificity of PCR reactions was confirmed by melting points analysis of PCR products. Realplex software (Eppendorf, Hamburg, Germany) was used for data acquisition and analysis. Correction for PCR performance as well as quantification relative to housekeeping gene Hmbs was carried out as described previously (Pfaffl, 2001 (link)).