title: GSM8801138: ExE_Dox_2; Mus; RNA-Seq
library_construction_protocol: Extra-embryonic tissues were dissected in PBS and collected in 150µl of 1x RNA Protection Reagent (NEB #T2011-1), snap-frozen and stored at -70C. RNA extraction was performed using the Monarch Total RNA Miniprep Kit (T2010S, NEB) following the protocol for mammalian whole blood RNA extraction with a few modifications. RNA was eluted in 30µl of water. Illumina libraries were prepared by using a modified smart-seq2 protocol [Picelli 2014] using SuperScript IV RT and tagmentation procedure as previously described [Henning 2018]. The RT reaction mix was as follows: 2 μl SSRT IV 5x buffer; 0.5 μl 100 mM DTT; 2 μl 5 M betaine; 0.1 μl 1 M MgCl2; 0.25 μl 40 U/μl RNAse inhibitor; 0.25 μl SSRT IV; 0.1 μl 100 uM TSO, 1.15 μl RNase-free H2O. RT thermal conditions: 52 °C 15 min, 80 °C 10 min. cDNA was generated using 16 PCR cycles. The cDNA cleanup (0.6x SPRI ratio) was carried out omitting the ethanol wash steps and the elution volume was 13 μl of H2O. For tagmentation, the sample input was normalized to 0.2 ng/μl. Obtained libraries that passed the QC step were pooled in equimolar amounts. Libraries were sequenced at 50 bp paired-end on NextSeq 2000 platform.