GSM3143575: Definitive differentiated rep3; Mus musculus; RNA-Seq
RNA was extracted from sorted EVP, TA and D cells using a QIAGEN mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA quality and concentration was assessed using A260nm/A280nm spectroscopy on the Nanodrop ND-1000 (Thermo-scientific, Langenselbold, Germany). 5-100ng of RNA was used for cDNA synthesis using the Superscript III Reverse Transcription Kit (Invitrogen, Mount Waverley, Australia). RNA-Seq libraries were prepared from purified total RNA using a modified Smart-Seq2 protocol developed by Picelli et al. 2 ng of purified total RNA (0.4 ng/µL) was combined with 1 µL of 10 µM oligo-dT primer (/5Biosg/AAGCAGTGGTATCAACGCAGAGTACT30VN; Integrated DNA Technologies) and 1 µL of dNTP mix (10 mM each; Invitrogen, y02256), then the protocol was continued as described. Briefly, the RNA was reverse transcribed with the Smart-Seq2 TSO (/5Biosg/AAGCAGTGGTATCAACGCAGAGTACATrGrGrG, Integrated DNA Technologies), followed by 12 cycles of PCR amplification to obtain enough cDNA to prepare a library. Volumes of reagents were scaled accordingly to maintain final concentration ratios as in the original protocol, except for the PCR preamplification where the Smart-Seq2 ISPCR primer (/5Biosg/AAGCAGTGGTATCAACGCAGAGT; Integrated DNA Technologies) was added to a final concentration of 0.25 µM. 0.5 ng of cDNA was prepped into a library using the Nextera XT DNA sample preparation kit (Illumina, FC-131-1096), with 12 cycles of PCR used to amplify the final library. The final Nextera XT libraries were quantified on the Agilent Bioanalyzer with the High Sensitivity DNA kit (Agilent Technologies,  4067-4626). Libraries were pooled in equimolar ratios, and the pool was quantified by qPCR using the KAPA Library Quantification Kit - Illumina/Universal (KAPA Biosystems, KK4824) in combination with the Life Technologies Viia 7 real time PCR instrument. Sequencing was performed using the Illumina NextSeq500 (NextSeq control software v2.0.2/ Real Time Analysis v2.4.11). The library pool was diluted and denatured according to the standard NextSeq protocol (Document # 15048776 v02), and sequenced to generate paired-end 76 bp reads using a 150 cycle NextSeq500/550 High Output reagent Kit v2 (Catalog # FC-404-2002).
GEO Accession
GSM3143575