TRAP: Translated and transcribed RNA from brain (forebrain and part of the diencephalon), head (without brain), heart, kidney, limb, liver or lung was purified from one to six mCherryTRAP embryos (age-dependent) as described (Hupe et al.: Nucleic Acids Res, 2014). HUVEC: RNA was purified using RNeasy® Micro Kit (Qiagen) following the manufacturer’s instructions. Single cell: Forebrains of mCherryTRAP E14.5 embryos were dissected in ice-cold PBS. The tissue was diced with a scalpel and subsequently enzymatically dissociated in PBS containing 1 mg/ml collagenase typ II (Worthington) and 1 µl/ml DNase I (Roche) at 37°C for 30 min with gentle sequential trituration. The cell suspension was filtered through a 40 µm cell strainer and 20 ml of DMEM containing 10 % FBS was added. After centrifugation at 800 x g for 5 min, the cell pellet was resuspended in 2 ml PBS containing 2 % FBS. Cells were sorted on a BD FACSAria III Cell Sorting system (BD Bioscience) based on mCherry fluorescence utilizing the 488-nm laser. mCherryTRAP-negative embryos were used to define background fluorescence. Forward and side scatter analysis were used as gates to avoid cell debris and doublets. Single cells were collected in a 96 well plate contain 4 µl of a mild hypotonic lysis buffer [0.1% Triton X-100 (Sigma), 1 U/µl recombinant RNase inhibitor (Promega), 2.5 µM anchored oligo-dT primer (5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′, Biomers), and 2.5 mM dNTP mix (Fermentas)] in each well. TRAP and HUVEC: RNA libraries were prepared for sequencing using standard Illumina protocols Single cell: Libraries were generated following Smart-seq2 (Picelli et al.: Nat Methods, 2013) and Tn5 transposase tagmentation (Picelli et al.: Genome Res, 2014) protocols.
GEO Accession
GSM2091303