The following fly strains were used: w1118, w-; stauD3 cn sp / Cyo; GFP-Stau2.2FL / TM3 and TM6B. All stocks were maintained at 25C.
Extracted molecule
total RNA
Extraction protocol
For RIPs with synthetic anti-Staufen antibody and synthetic control antibody, synthetic antibodies were expressed and purified as Fabs, as described (Laver et al. 2012). Extract was prepared from embryos collected 0-to-3 hours post-egg-laying and immunoprecipitations were performed as described (Laver et al. 2012), with the following modifications: RNA co-immunoprecipitations were performed using approximately 800 µL of ½ diluted embryo extract (see Laver et al. 2012), extract was incubated with 80 µL of anti-FLAG M2 affinity gel (Sigma) carrying 40 µg of purified FLAG-tagged Fab and blocked with BSA. Fabs with associated protein and RNA were eluted from the beads by three 20 minute elutions at 4˚C with 100 µL of FLAG peptide (Sigma), the three eluates were pooled, and RNA was isolated from the eluate using TRI Reagent (Sigma). For immunoprecipitation of GFP-Staufen, extract was prepared from 0-to-3 hr embryos as described (Laver et al. 2012), but using a different protease inhibitor cocktail (Roche CAT#11836170001). Extracts were thawed and pooled to create a single replicate of 600-800µL and Triton X-100 was added to a final concentration of 0.1%. A 10µL aliquot of the extract was used to isolate RNA for the input sample (using Invitrogen’s TRIzol reagent, according the manufacturer’s protocol). The remaining extract was divided in half for use in the anti-GFP and control anti-FLAG immunoprecipitations. For anti-GFP-Staufen immunoprecipitations, protein G magnetic beads were first blocked using a protocol adapted from Ren et al. (Ren et al. 2000): two aliquots of 100 µL of beads (Invitrogen Dynabeads® Protein G CAT#100.04D) were each washed 3 times with 1-1.5 mL of 5 mg/mL PBS/BSA, then blocked overnight in 250µL PBS/BSA at 4C. The immunoprecipitations were then performed using the following protocol, adapted from Invitrogen’s Dynabeads® Protein G protocol and Roche’s immunoprecipitation protocol for anti-GFP (CAT#11814460001), was used: one aliquot of 100µL of beads that was blocked overnight was washed three times with 1.5mL of PBS/BSA and the beads were then divided into two new tubes. Each aliquot of beads was resuspended in an equal volume of embryo extract (300-400µL) and the tubes rotated at 4oC for 1hr. The samples of cleared extract were then transferred to new tubes and 2µg of anti-GFP or anti-FLAG (Sigma-Aldrich CAT#F3165) antibody added to each, for the anti-GFP and mock immunoprecipitations respectively. The samples were then mixed and incubated on ice at 4oC for 1hr. The remaining 100µL aliquot of blocked beads was washed three times with 1.5mL of lysis buffer 150 (150 mM KCl, 20 mM Hepes pH 7.4, 1 mM MgCl2) supplemented with 0.1% Triton X-100. The beads were split into two new tubes, the anti-GFP extract being added to one and the anti-FLAG extract to the other, and each incubated with rotation at 4oC for four hours. The depleted extract was then removed and the beads washed three times with 200µL of lysis buffer 150 with Triton. Finally, the beads were transferred to new tubes where they were resuspended in 500µL of TRIzol for RNA purification according to the manufacturer’s protocol. The RNA retrieved from these extractions was purified and concentrated using the kit and procedure from Zymo Research (CAT#R1015). Total RNA was extracted by TRIzol according to the manufacturer’s protocol.
Label
Cy5
Label protocol
Labelling was performed following the protocol described in the NimbleGen Array User’s Guide (Gene Expression Arrays, version 5.0).
Hybridization protocol
Hybridization was performed following the protocol described in the NimbleGen Array User’s Guide (Gene Expression Arrays, version 5.0).
Scan protocol
Arrays were scanned with a GenePix4000B microarray scanner system (Molecular Devices, Inc., Sunnyvale, CA, USA), and scanned images were initially quantified using Nimblescan (Roche), following the protocol described in the NimbleGen Array User's Guide (Gene Expression Arrays, version 5.0).
Description
This sample is total RNA isolated from 0-3 hour GFP-Staufen transgenic embryos. It is the first of three biological replicates. SAMPLE 10
Data processing
The raw data (.pair files) were normalized with ArrayStar 3 (DNASTAR) software using the RMA quantile method, and signal intensities were log2-transformed. For normalization, samples were grouped as follows, with each group normalized separately: Samples 1-3, Samples 4-9, Samples 10-12, Samples 13-18.
