title: GSM2984588: L16367_M4-1m_07_E10; Mus musculus; RNA-Seq
library_construction_protocol: To isolate Aldh1b1 expressing cells and their progeny, 1 mg of corn oil dissolved tamoxifen (20 mg/ml; Sigma) was injected intraperitoneally in Aldh1b1CreERT2 / ROSA26 LSLtdTomato double heterozygous adult mice for 5 consecutive days. Aldh1b1 expressing cells and their early progeny were isolated one day later from pancreata dissociated into single cells by virtue of their tdTomato (PE-Texas Red channel) expression from the lineage tracing mouse pancreas by FACS.  Pancreata were dissociated into single cells and cells were FACsorted into a 96 well plate containing 2 ul of nuclease free water with 0.2% Triton-X 100 and 4 U murine RNase Inhibitor (NEB), spun down and frozen at -80°C. Lysis, reverse transcription of the RNA and cDNA amplification was performed according to the smartseq-2 protocol 6 with few modifications as detailed below. After thawing the samples, 2 ul of a primer mix (5 mM dNTP (Invitrogen), 0.5 uM dT-primer*, 4 U RNase Inhibitor (NEB)) was added. After filling up to 10 ul with RT buffer mix for a final concentration of 1x superscript II buffer (Invitrogen), 1 M betaine, 5 mM DTT, 6 mM MgCl2, 1 uM TSO-primer*, 9 U RNase Inhibitor and 90 U Superscript II, RNA was denatured for 3 minutes at 72°C and reverse transcription was performed at 42°C for 90 min. The reverse transcriptase was then inactivated at 70°C for 15 min. The cDNA was amplified using Kapa HiFi HotStart Readymix (Peqlab) at a final 1x concentration and 0.1 uM UP primer* under the following cycling conditions: initial denaturation at 98°C for 3 min, 22 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 hydrophobic Sera-Mag SpeedBeads (GE Healthcare) and the DNA was eluted in 12 ul nuclease free water. The concentration of the samples was measured with a Tecan plate reader Infinite 200 pro in 384 well black flat bottom low volume plates (Corning) using AccuBlue Broad range chemistry (Biotium). For library preparation 700 pg cDNA in 2 ul was mixed with 0.5 ul tagment DNA enzyme and 2.5 ul tagment DNA buffer (Nextera, Illumina) and tagmented at 55°C for 5 min. Subsequently, Illumina indices were added during PCR (72°C 3 min, 98°C 30 sec, 12 cycles [98°C 10 sec, 63°C 20 sec, 72°C 1 min], 72°C 5 min) with 1x concentrated KAPA Hifi HotStart Ready Mix and 0.7 uM dual indexing primers. After PCR, libraries were quantified with AccuBlue Broad range chemistry, equimolarly pooled and purified twice with 1x volume Sera-Mag SpeedBeads. This was followed by Illumina sequencing on a Nextseq500 aiming at an average sequencing depth of 0.5 mio reads per cell. Primers were as follows: dT primer: Aminolinker-AAGCAGTGGTATCAACGCAGAGTCGACT(30)VN; Template Switch Oligo (TSO) primer: AAGCAGTGGTATCAACGCAGAGTACATrGrGrG; Universal Part (UP) primer: AAGCAGTGGTATCAACGCAGAGT.
attributes.tag: GEO Accession
attributes.value: GSM2984588