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Sample GSM3265596

Query DataSets for GSM3265596

Status

Public on Aug 05, 2018

Title

PB_1_d1_rep1

Sample type

SRA

Source name

Protocerebral bridge PB_1

Organism

Drosophila melanogaster

Characteristics

tissue: head
genetic driver: PB_1_d1
driver type: anatomicalCellType:single
external driver id: SS00116
intact reporter: 10XUAS_UNC84_attP2
intact protocol: TAPIN

Growth protocol

Split-GAL4 and GAL4 driver lines were used to express the INTACT reporter (UNC84-2XGFP) in defined cell populations

Extracted molecule

nuclear 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
s129

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