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Sample GSM3265542 Query DataSets for GSM3265542
Status Public on Aug 05, 2018
Title opticlobe_d2_rep2
Sample type SRA
 
Source name dissected opticlobe after lamina removal
Organism Drosophila melanogaster
Characteristics tissue: dissected opticlobe after lamina removal
genetic driver: opticlobe_d2
driver type: dissected
external driver id: SS00796
intact reporter: NA
intact protocol: bulk
Growth protocol Split-GAL4 and GAL4 driver lines were used to express the INTACT reporter (UNC84-2XGFP) in defined cell populations
Extracted molecule total RNA
Extraction protocol INTACT protocol: Adult flies were entrained to a 12:12 light:dark cycle, anesthetized by CO2 at ZT8 - ZT12, flash frozen in liquid N2 and decapitated by vigorous vortexing. Heads or wings/appendages were then collected on cooled metal sieves (H&C Sieving Systems: 1296, 1297, 1298, 1301). Both flies and purified frozen material can be stored indefinitely at -80degrees C. In a typical experiment 100 to 500 frozen heads were added to 5mls of 20mM Beta-glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40, 0.5mM spermidine, 0.15mM spermine, 1mM DTT, 1X complete protease inhibitor (Sigma: 5056489001), 3mg/ml BSA (ThermoFisher: AM2618), 1mg/ml torula RNA (ThermoFisher: AM7118), 0.6mg/ml carboxyl coated Dynabeads (ThermoFisher: 14306D) and 2 microgram anti-GFP antibody (ThermoFisher: G10362). Homogenization was carried out on ice by 50 tractions in a Dounce homogenizer using the tight pestle followed by filtration over a 10 micrometer cup filter (Partec: 0400422314). Released chromatin and broken nuclei were adsorbed to carboxyl coated magnetic beads for 30 minutes at 4 degrees C with constant rotation. After which the beads were removed on a magnetic stand, the supernatant diluted to 50mls with 20mM Beta-glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40, 0.5mM spermidine, 0.15mM spermine, 1mM DTT and 1X complete protease inhibitor (Sigma: 5056489001), filtered over a 1 micrometer cup filter (Pluriselect: 435000103) and split into two equal volumes. A 40% Optiprep (Sigma: D1556), 20mM Beta-glycerophosphate pH7, 2mM EDTA and 0.5% NP40 solution was then carefully placed under each aliquot. Followed by a lower layer of 50% Optiprep, 20mM Beta-glycerophosphate pH7, 2mM EDTA and 0.5% NP40. Nuclei were then pelleted on to the 50% layer for 30 minutes at 2300Xg. Purified nuclei were passed over a 10 micrometer cup filter, diluted to 10mls with 20mM Beta-glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40, 0.5mM spermidine, 0.15mM spermine, 1mM DTT and 1X complete protease inhibitor and incubated with 30 microliters of protein G Dynabeads (ThermoFisher: 10004D) for 40 minutes on ice with occasional agitation. Bead-bound nuclei were recovered on a magnet stand followed by a 20 minute incubation on ice in 9mls of 20mM Beta-glycerophosphate pH7, 300mM NaCl, 1M urea, 0.5% NP40, 2mM EDTA, 0.5mM spermidine, 0.15mM spermine, 1mM DTT, 1X complete protease inhibitor, 0.075mg/ml torula RNA and 0.05Units/ml Superasin (ThermoFisher: AM2696). Nuclei were then recovered on a magnet stand, resuspended in 1ml of the previous buffer, passed over a 10 micrometer cup filter, a 5 microliter aliquot was withdrawn for quantitation and the remainder of the sample solubilized in Arcturus Picopure RNA extraction buffer (ThermoFisher: KIT0204).
TAPIN-seq protocol: 100 to 3000 frozen heads were added to 5mls of sodium acetate ph8.5, 2.5mM MgCl2, 250mM sucrose, 0.5% NP-40, 0.6mM spermidine, 0.2mM spermine, 1mM DTT, 1X complete protease inhibitor, 0.5mg/ml torula RNA, 0.6mg/ml carboxyl coated Dynabeads and 2microgram anti-GFP antibody. Homogenization was carried out on ice by 50 tractions in a Dounce homogenizer using the tight pestle followed by filtration over either a 10 or 20 micrometer cup filter (Partec: 0400422314, 040042315). Released chromatin and broken nuclei were adsorbed to carboxyl coated magnetic beads for 30 minutes at 4 degrees C with constant rotation. Unbound antibody was removed by incubating the sample on ice for 20 minutes with 100 microliters of washed UNOsphere SUPra resin (Biorad: 1560218). After the resin was removed on a 10 micrometer cup filter and the carboxyl beads on a magnet stand, the nuclei-containing supernatant was mixed with an equal volume of 500mM sodium acetate ph8.5, 250mM sucrose, 6mM EGTA, 6mM EDTA, 0.6mM spermidine, 0.2mM spermidine, 1mM DTT, 1X complete protease inhibitor, 0.25mg/ml torula RNA and 30 microliters Protein A Dynabeads (ThermoFisher: 10002D). A 2 hour incubation on ice with occasional agitation was used to recover tagged nuclei. Bead-bound nuclei were then recovered on a magnet stand and washed twice with 250mM sodium acetate ph8.5, 250mM sucrose and 0.1% NP40. Nuclei were then released at 37 degrees C for 1 hour by incubation in 50 microliters of 10mM Tris pH7.5, 2.5mM MgCl2, 0.5mM CaCl2, 250mM sucrose, 0.1% NP40, 1mg/ml torula RNA, 40 units RNAsin (Promega: N2515), 2 units DNAseI (NEB: M0303L), 320 units IdeZ protease (NEB: P0770S). The sample was diluted to 100 microliters with 10mM Tris pH7.5, 2.5mM MgCl2, 0.5mM CaCl2, 250mM sucrose and 0.1% NP40, EGTA was added to 1mM and the suspension was rapidly triturated 100 times. After returning the sample to a magnet stand, 90 microliters of buffer containing released nuclei was removed and added to 1.5 microliters of Protein G Dynabeads that were previously resuspended in 10 microliters of 10mM Tris pH7.5, 2.5mM MgCl2, 0.5mM CaCl2, 250mM sucrose and 0.1% NP40. The second binding reaction was run for 1 to 3 hours on ice with occasional agitation, followed by 2 X 250 microliters washes in 10mM Tris pH7.5, 2.5mM MgCl2, 0.5mM CaCl2, 250mM sucrose and 0.1% NP40. Prior to the last wash a 5 microliter aliquot was removed for quantitation and the remainder of the sample was solubilized in Arcturus Picopure RNA extraction buffer.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description QC: pass
s075
Data processing We trimmed five nucleotides from the 5’ end of reads using seqtk (https://github.com/lh3/seqtk) to remove potential contaminating adapter sequence from the NuGen Ovation kit. We estimated the abundance of annotated genes using kallisto (v 0.43.1; Bray et al., 2015) to pseudo-align trimmed reads to the fly transcriptome (cDNA and ncRNA transcript sequences from ENSEMBL release 91, based on FlyBase release 2017_04), ERCC spike-ins, and the INTACT construct sequences GAL4-DBD, p65-AD, and UNC84_2XGFP.
ERCC, INTACT tag constructs, and rRNA genes were removed from the abundance tables and the estimated abundances of the remaining genes were renormalized to one million total transcripts.
We further analyzed high-quality libraries passing three criteria: > 8,500 genes detected, > 3ug cDNA yield, r>= 0.85 pearson correlation between replicates
We modeled transcript abundance (in high-quality libraries) as arising from a gene-specific mixture of on and off log-normal components (See manuscript for details). We used this model to transform transcript abundance into probabilities of expression (probability of arising from on state).
Genome_build: dm6. ENSEMBL BDGP.r91
 
Submission date Jul 11, 2018
Last update date Aug 05, 2018
Contact name Fred P. Davis
E-mail(s) Fred.davis@nih.gov
Organization name NIH
Department NIAMS
Street address 9000 Rockville Pike
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platform ID GPL17275
Series (1)
GSE116969 A GENETIC, GENOMIC, AND COMPUTATIONAL RESOURCE FOR EXPLORING NEURAL CIRCUIT FUNCTION
Relations
BioSample SAMN09650005
SRA SRX4384113

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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