title: CAGE_meso_S13_r1
organism: Drosophila melanogaster
description: Protocols: Flies expressing EGFP-CBP20 under control of a mesodermal enhancer were grown in population cages at 25 degrees. After three/four 1 hr pre-lays, the flies were allowed to lay for 1 or 2 hrs, after which the embryos were aged at the same temperature to the appropriate time-point. The embryos were then dochorionated using 50% bleach, washed with distillate water and PBT (PBS + 1% Triton X-100), and directly subjected to homogenization on ice. All steps were done at 4C. 500 ul of embryo suspension was added to 6.5 ml Schneider???s media with Actinomycin D in a 15 ml dounce homogenizer on ice and dounced with a loose pestle 7 times, in aliquots of less than 0.0625 g of embryos. Material from two douncing rounds was combined in one 15 ml tube and centrifuged at 600 rpm for 5 min at 4C. Supernatant was transferred into a clean tube and centrifuged at 1700 rpm for 10 min at 4C. Supernatant was discarded, and 250 ul of Schneider???s media complemented with 8% fetal bovine serum and 1 ug/ml of Actinomycin D was added to the pellet. All resuspended pellets were combined into a single tube, cells were gently passed through a 18-gauge needle 5 times and sieved through a 40 um sieve into a 50 ml tube. Approximately 5% of the total sample was transferred into a RNAse free 1.5ml tube and centrifuged at 800 g for 10 min at 4C. 600 ul TRIzol was added to the pellet and saved as unsorted sample. Remaining sample was used for fluorescence activated cell sorting (FACS). Cell sorter (MoFlo, Beckman Coulter) was precooled at 4C and run with 70 um nozzle and 5,000 ??? 10,000 cells per second. Sorted cells were collected in 5 ml round bottom polypropylene tubes in 500 ul Seecof saline (6 mM Na2HPO4, 3.67 mM KH2PO4, 106 mM NaCl, 26.8 mM KCl, 6.4 mM MgCl2, 2.25 mM CaCl2, pH 6.8) supplemented with 0.1 U/ul RNAse inhibitor. Sorted cells were aliquoted in low binding RNAse free tubes and centrifuged at 800 g 10 min at 4C. Pellet was resuspended in 150 ul TRIzol LS and all samples were pooled into a single tube before proceeding with RNA isolation. RNA isolation was done according to manufacturer???s instructions for TRIzol LS, including overnight precipitation step with 1 ul glycogen at -80C. RNA was treated with RNAse free DNAse I (Roche) for 30 min and purified with Agencourt RNAclean XP beads (Beckam Coulter) according to manufacturer???s instructions. 'U' extracts: RNA extracted from cells before sorting (see below) 'S' extracts: RNA extracted from sorted cells. Production of libraries containing 27bp-tags from 5' ends of capped transcripts. Taken from Takahashi et al, Nat Prot 2012. Briefly, 1-5ug of total RNA are retrotranscribed with a primer containing a common sequence followed by 15 Ns. The RNA-cDNA duplexed is subjected to in-vitro biotinylation of the 5'cap. Then the single-stranded RNA is degraded with RNaseI and the remaining biotinylated (capped) RNA-cDNA duplexes are pull-down with streptavidin-conjugated Dynabeads. cDNA is released through NaOH treatment, and then a barcoded 5'adapter is ligated to the 3'end of the single strand cDNA. Non-ligated adapter is removed by purifying twice with AMPure XP beads. Second strand is synthesized using a primer corresponding to the 5'adapter. Then, 27bp-long tags are generated by digestion with EcoP15I, a type III restriction enzyme. The two matched restriction sites are in the 5'adapter and in the RT primer sequences. A common 3'adapter is then ligated to the resulting fragments. The libraries are amplified by PCR, tipically for 8-15 cycles. After PCR, primers are removed with exonuclease treatment, and the double strand fragments are purified both with QIAquick columns and AMPure XP beads to remove unwanted adapter dimers.