GSM4760856: A471_het_P; Mus musculus; RNA-Seq
At 4 and 10 days after the induction of mutagenesis, intestinal crypts were dissociated into single cells with TrypLE Express (Thermo Fisher Scientific) for 30min at 37°C. Dissociated cells were passed through 70µm cell strainer and washed wit 5% FBS/PBS. Cells were stained with PE-conjugated anti-CD24,  APC-conjugated anti-CD326 (EpCAM) antibody and Alexa-Fluor 700 CD45 antibodies for 45min on ice. Stained cells were collected by centrifugation and resuspended in SYTOX blue dead cell stain. FACS sorting was performed on a FACS AriaTM III cell sorter (BD). Cells are FACsorted into a PCR tube 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. After thawing the samples, RNA is denatured for 3 minutes at 72 °C in the presence of 2.4 mM dNTP (Invitrogen), 240 nM dT-primer* and 4 U RNase Inhibitor (NEB). The reverse transcription and addition of the template switch oligo is 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*, 9 U RNase Inhibitor and 90 U Superscript II. The reverse transcriptase is inactivated at 70 °C for 15 min. The single stranded cDNA is subsequently amplified using Kapa HiFi HotStart Readymix (Roche) at a 1x concentration together with 250 nM UP-primer* under following cycling conditions: initial denaturation at 98°C for 3 min, 16 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 is 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 is determined with the Fragment Analyzer (Agilent). For library preparation 1.5 ng cDNA was tagmented using 0.5 µl TruePrep Tagment Enzyme V50 and 1x TruePrep Tagment Buffer L (TruePrep DNA Library Prep Kit V2 for Illumina, Vazyme), followed by an incubation step at 55 °C for 10 min. Next, Illumina indices are 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 300 nM dual indexing primers. After PCR, libraries are purified twice with 1x volume Sera-Mag SpeedBeads and quantified with the Fragment Analyzer, followed by Illumina sequencing on a Nextseq500 with a sample sequencing depth of 30 mio reads on average.
GEO Accession
GSM4760856