title: Twist::EGFP-CBP20
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. Total RNA was depleted from rRNA, using an strategy based on Khodor et al, Genes&Dev 2011. Briefly, 32 biotinylated oligos annealing Drosophila rRNA were used for retrotranscription of 1-5ug of total RNA, and the RNA-cDNA duplex was pulled-down with streptavidin-conjugated Dynabeads. The removal was performed 2 consecutive times. Then, RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (protocol B). Barcoded Y-shaped adapters were used for ligation, and standard common PCR primers for Illumina were used for amplification, tipically 14-15 cycles.
attributes.tag: ENA first public
attributes.value: 2018-10-24
attributes.tag: ENA last update
attributes.value: 2015-11-25
attributes.tag: External Id
attributes.value: SAMEA3674405
attributes.tag: INSDC center alias
attributes.value: EMBL
attributes.tag: INSDC center name
attributes.value: European Molecular Biology Laboratory
attributes.tag: INSDC first public
attributes.value: 2018-10-24T17:03:08Z
attributes.tag: INSDC last update
attributes.value: 2015-11-25T10:29:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4069:Twist::EGFP-CBP20
attributes.tag: age
attributes.value: 4-6h
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fruit fly
attributes.tag: developmental stage
attributes.value: stage 8-9
attributes.tag: initial time point
attributes.value: egg laying
attributes.tag: organism part
attributes.value: Mesodermal cells
attributes.tag: sample name
attributes.value: E-MTAB-4069:Twist::EGFP-CBP20
attributes.tag: strain
attributes.value: Twist::EGFP-CBP20