title: GSM4808739: WT.3.hdm; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated using Rneasy Micro Kit (Qiagen) and eluted in nuclease-free water. RNA was desiccated to 3 µl and 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). 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*, 9 U RNase Inhibitor and 90 U Superscript II. The reverse transcriptase was inactivated at 70 °C for 15 min and single stranded cDNA was 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, 10 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 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 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 300 nM dual indexing primers. After PCR, libraries were 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.