Illumina HiSeq 2000 sequencing; GSM1905878: ED1004_25163_FEMALE_rep1; Drosophila melanogaster; RNA-Seq

ED1004_25163_FEMALE_rep1

In order to understand the effect of genetic background on the response to gene dose perturbation, we performed mRNA transcriptional profiling on 99 hemizygotic lines (Df/+) from the DrosDel project, which have hybrid genetic background of OregonR/w1118. Overall design: We performed RNA-Seq analysis of 417 single adult flies in duplicate or triplicate.  Flies are from 73 different genotypes. Differential gene expression was analyzed separately for each sex, gene expression from each genotype was compared to normalized mean of gene expression remaining 72 genotypes.

Expression profiling individual DrosDel flies heterozygous for deletions of chromosome 2L in a hybrid background

Submitted by Gene Expression Omnibus on 01-FEB-2016

Single, day 3-5 adult male or female flies were partially crushed, stored in 100 ul of RNAlater (Life Technologies, Grand Island, NY), and frozen at - 80 oC for long term storage until RNA preparation. We used Mini-BeadBeater 96 (Biospec Products, Bartlesville, OK) for the homogenization of flies. Approximately 100 ul of 1 mm glass beads (Biospec Products) were added to the flies in RNAlater. All samples had the same bead beating conditions which were 3 x 1 minute with a 2 minute rest on ice between each beat beating, which was in 1 ml Axygen 96 well plate (Corning, Union City, CA). For RNA isolation, we added 600ul of RLT buffer (Qiagen, Valencia, CA) to each well to dilute RNAlater solution. Total RNA was isolated with RNeasy 96 kit (Qiagen) as in manufacturer’s handbook (Protocol for Isolation of Total RNA from Animal Cells using spin technology, Cat#19504). The amount of total RNA extract was measured using Quant-iT RiboGreen (Life Technologies). 400 ng of total RNA in 50 ul of nuclease-free water was mixed with 50 ul of 2:5 dilution of Dynabeads Oligo(dT)25 (Life Technologies) that has been rinsed and diluted with Binding Buffer (20 mM Tris-HCl pH7.5, 1.0 M LiCl, 2 mM EDTA). The mixture was heated to 65 oC for 5min in a thermocycler, and cooled down on ice for 1 min. After 15 min of incubation at room temperature, the beads were collected with a magnetic stand, and rinsed with 200 ul Washing Buffer (10 mM Tris-HCl pH7.5, 0.15 M LiCl, 1mM EDTA) for 1 min at 1,000 rpm (Thermomixer, Eppendorf, Hauppauge, NY). The beads were collected again with a magnetic stand, and subjected to elution with 50 ul of Elution buffer (10 mM Tris-HCl pH7.5) at 80 oC for 2 min. The elute were rebound to the beads by incubating with 50 ul Binding buffer, and rinsed with 200 ul Washing buffer as above. The poly A+ RNA on beads were eluted and fragmented with 16 ul of Fragmentation buffer that contained 1:4 dilution of 5X First Strand Buffer from Protoscript II (New England BioLabs, Ipswich, MA), 500 ng of random primers (Life Technologies), and 20 pg of ERCC spike-ins Pool 78A or 78B (Zook et al., 2012) at 94 oC for 6 min. The beads were removed using a magnetic stand, and 10 units of SuperRase-in (Life Technologies), 100 unit of Protoscript II reverse transcriptase, DTT (New England BioLabs, 5 mM final) and dNTPs (Enzymatics, Beverly, MA. 625 uM final) were added for reverse transcription. Thermocycler was set as 25 oC for 10 min - 42 oC for 50 min - 70 oC for 15 min - 4 min Hold. The DNA-RNA hybrids were cleaned up with  1.9 volume of MagNA beads (Rohland and Reich, 2012), mixed with 0.85 volume of ethanol. For that, 15 min of incubation was performed for binding of DNA-RNA hybrid to the beads at room temperature. The beads were sit on a magnetic stands to remove the liquid, and rinsed with 200 ul of 80% ethanol twice. After 5 min of air-dry, the beads were eluted with 16 ul of Elution buffer. For the second strand synthesis, 2.5 units of RNase H (Enzymatics) and 10 units of DNA polymerase I (Enzymatics) were added. The reaction was made on 16 oC for 5 hours with Blue buffer (Enzymatics, 1X final), DTT (10 mM final), dATP, dCTP, dGTP (Enzymatics, 0.5 mM final) and dUTP (ThermoFisher Scientific, Waltham, MA. 1 mM final) for a strand-specific protocol. The DNA products were cleaned up with MagNA beads and eluted as above but the beads were maintained. Purified double-stranded DNA was subjected to end-repair with NEBNext End Repair Module (New England BioLabs) as manufacturer’s protocol in 20 ul size. The para-magnetic beads were replenished with 1.9 volume of XP buffer (Wang et al., 2011), and cleaned up as like MagNA beads steps. For adapter ligation, adenylation was performed on blunt-ended DNA. 2.5 units of Klenow 3’-5’ exo (Enzymatics) was added to DNA. The mixture was incubated at 37 oC for 30 min with 0.5 mM (final) and 1X Blue buffer (Enzymatics). 1.9 volume of XP buffer was used to clean up as above. The DNA was eluted with 10 ul of Elution buffer,  and 1 ul of 24 differently barcoded adapters from TruSeq v2 kit (Illumina, San Diego, CA) were added for ligation. 24 ul reaction was set up with T4 DNA ligase (Rapid) (Enzymatics) for 20 min at 20 oC. The ligation was stopped by adding ⅓ volume of 0.03M EDTA, and the result was clean-up with 24 ul of XP buffer as above. After eluting with 30 ul Elution buffer, the DNA was cleaned up again with 1 volume of XP buffer to make 12 ul ligated products. dUTP incorporated strands of DNA were digested with 5 units of Uracil DNA Glycosylase (New England BioLabs) for 30 min at 37 oC. Then the DNA was amplified with 1.5 ul of P5 and P7 primers (Sequence from Illumina. ordered from Integrated DNA Technology, Coralville, Iowa) with KAPA HiFi HotStart DNA polymerase (KAPA biosystems, Wilmington, MA) in 30 ul scale. The PCR parameters were 98 oC for 45 sec, 14 rounds of 98 oC for 15 sec - 60 oC for 30 sec - 72 oC for 30 sec, and 72 oC for 5 in. The amplified DNA was cleaned up with MagNA beads. The fully made libraries were quantified with Quant-iT PicoGreen (Life Technologies), and pooled to be sequenced in Illumina HiSeq 2000 (Illumina).

Expression profiling individual DrosDel flies heterozygous for deletions of chromosome 2L in a hybrid background

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