RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster
- University of California, Berkeley, United States
- Lawrence Berkeley National Laboratory, United States
- University of California, Irvine, United States
- National University of Ireland, Ireland
Figures
Figure 1 with 6 supplements
AAGAG(n) RNA localizations in embryos and larvae.
(a) AAGAG RNA distributions (magenta) throughout embryonic and larval development in Oregon R flies. DNA/DAPI = blue; all images are confocal sections. White box indicates location of enlarged …
Figure 1—figure supplement 1
RNA-FISH analysis of satellite RNAs in cycle 14 embryos.
(a) ND = Not detected, *repeats tested for expression from both strands. For more details about AAGAG RNA in cycle 14, see Figure 1, A and E and Figure 1—figure supplement 2D. (b) Projections of …
Figure 1—figure supplement 2
AAGAG RNA is present throughout development and forms foci.
(a) Northern blot analyzing RNA from Oregon R embryos and third instar larva hybridized with a probe complementary to AAGAG(n). Note that signal intensity is not a representation of relative AAGAG …
Figure 1—figure supplement 3
AAGAG RNA foci contain single-stranded RNA and are not associated with R-loops.
Confocal sections of embryonic nuclei in cycle 14 (with exception of left panel in ‘b’), nuclear periphery outlined in dotted circles. (a) No RNase control. (b) Treated with RNaseIII (left nucleus …
Figure 1—figure supplement 4
AAGAG RNA transcripts originate from 2R, X and 3R heterochromatin loci and are transcribed in embryos and larval brain.
(a) Unique regions adjacent to AAGAG(n), or AAGAG(n) within AG rich regions, were identified as potential sources of satellite transcripts, as described in Materials and methods. (+) indicates …
Figure 1—figure supplement 5
AAGAG RNA-FISH localizes RNA and not DNA.
Confocal sections of cycle 14 nuclei treated with either (a) RNAseH or (b) RNAseIII after AAGAG RNA probe (magenta) hybridization. A higher laser intensity for the AAGAG probe channel was used in b …
Figure 1—figure supplement 6
AAGAG RNA is decreased and foci abolished in L3 with actin-GAL4-driven RNAi to AAGAG, without affecting levels of genes whose mRNAs contain short runs of AAGAG.
Also, AAGAG RNAi results in lethality. (a-c) Left column- section of brain lobes stained with DAPI (blue) and RNA-FISH to AAGAG RNA (magenta) imaged with the same intensity. Arrows point to AAGAG …
Figure 2 with 3 supplements
AAGAG RNA is enriched in primary spermatocytes and necessary for male fertility.
(a) Confocal section of a larval testis. RNA-FISH to AAGAG = magenta, H2Av (chromatin) IF = gray, DNA (DAPI) = blue. S3, S5, and S6 refer to primary spermatocyte stages. (b) Enlarged confocal …
Figure 2—figure supplement 1
Overview of normal spermatogenesis and defects observed after AAGAG RNA depletion.
(a) Spermatogenesis in Drosophila melanogaster initiates at the apical end of the testes (Hub), where GSCs divide asymmetrically, producing gonialblasts (GBs) that begin cell-differentiation. GB …
Figure 2—figure supplement 2
Heterochromatic regions adjacent to AAGAG(n) or AG(n)-rich blocks are transcribed in primary spermatocytes, co-localize with AAGAG(n) RNA foci and do not come from the Y.
(a) Projections of Oregon R S5 spermatocytes probed for unique regions of RNA (green) adjacent to AAGAG(n) (magenta) or AAGAG(n) containing AG rich blocks. DAPI (DNA) is indicated in blue. (b) …
Figure 2—figure supplement 3
AAGAG RNA and not CUCUU RNA is substantially decreased in Bam-GAL4- driven AAGAG RNAi, and AAGAG RNA levels are increased in rescue experiments.
(a) Although visibly absent in embryos and somatic larval tissues, CUCUU RNA (green) is expressed in adult spermatocytes. Note that CUCUU RNA is localized to the S5 lumen, internal to the chromatin …
Figure 3 with 1 supplement
AAGAG RNAi depletion in mitotic germline cysts and spermatocytes (Bam-GAL4 driver) results in severe defects in sperm maturation and protamine deposition.
(a) Seminal vesicles (SVs) in testes from 0 to 6 hr old adults; DAPI (DNA) = cyan. Mature sperm nuclei visible as thin, elongated DAPI signals in the scrambled control (top, white arrow) are absent …
Figure 3—figure supplement 1
Histones are retained and DNA morphology is altered in late canoe stage AAGAG RNAi testes.
(a) Histones are retained until late canoe stage with AAGAG RNAi in primary spermatocytes but removed by early canoe stage in scrambled RNAi. (b) Canoe stage nuclei exhibit abnormal morphology with …
Figure 4
Model for AAGAG RNA function during spermatogenesis.
AAGAG RNA (magenta) present only in primary spermatocytes (light blue = chromosome territories) acts directly or indirectly to promote important processes later in sperm maturation, including the …
Tables
Table 1
Male fertility in AAGAG RNAi with GAL4 drivers expressed at earlier testes stages than Bam.
| GAL4 RNAi driver | Expression location (Demarco et al., 2014) | % fertile | + /- stdev. | Minimum number of males per set |
|---|---|---|---|---|
| Fascillin | Hub | 94 | 16 | 15 |
| PTC | Soma- CySCs and cyst cells | 90 | 5 | 18 |
| Traffic Jam | Soma- Hub and CySCs | 97 | 4 | 12 |
| Dpp1 | Soma- CySCs and early cyst cells | 96 | 6 | 17 |
| Nanos | Germline- GSCs and early germline cysts | 83 | 5 | 13 |
Table 2
Oligos for RNA probes.
| Repeat or region | Oligo with T3 antisense promoter |
|---|---|
| CAGC(n) | CAGCCAGCCAGCCAGCCAGCCAGCTCTCCCTTTAGTGAGGGTTAATT |
| CCCA(n) | CCCACCCACCCACCCACCCACCCACCCATCTCCCTTTAGTGAGGGTTAATT |
| CATTA(n) | CATTACATTACATTACATTACATTATCTCCCTTTAGTGAGGGTTAATT |
| CGGAG(n) | CGGAGCGGAGCGGAGCGGAGCGGAGTCTCCCTTTAGTGAGGGTTAATT |
| CGA(n) | CGACGACGACGACGACGACGACGATCTCCCTTTAGTGAGGGTTAATT |
| CAACT(n) | CAACTCAACTCAACTCAACTCAACTTCTCCCTTTAGTGAGGGTTAATT |
| CGAAG(n) | CGAAGCGAAGCGAAGCGAAGCGAAGTCTCCCTTTAGTGAGGGTTAATT |
| CCCCAG(n) | CCCCAGCCCCAGCCCCAGCCCCAGTCTCCCTTTAGTGAGGGTTAATT |
| CCGAG(n) | CCGAGCCGAGCCGAGCCGAGCCGAGTCTCCCTTTAGTGAGGGTTAATT |
| CGGAA(n) | CGGAACGGAACGGAACGGAACGGAATCTCCCTTTAGTGAGGGTTAATT |
| CACCC(n) | CACCCCACCCCACCCCACCCCACCCTCTCCCTTTAGTGAGGGTTAATT |
| CTAGT(n) | CTAGTCTAGTCTAGTCTAGTCTAGTTCTCCCTTTAGTGAGGGTTAATT |
| CATCG(n) | CATCGCATCGCATCGCATCGCATCGTCTCCCTTTAGTGAGGGTTAATT |
| CAT(n) | CATCATCATCATCATCATCATCATTCTCCCTTTAGTGAGGGTTAATT |
| CAAAC(n) | CAAACCAAACCAAACCAAACCAAACTCTCCCTTTAGTGAGGGTTAATT |
| CGAAA(n) | CGAAACGAAACGAAACGAAACGAAATCTCCCTTTAGTGAGGGTTAATT |
| CATAT(n) | CATATCATATCATATCATATCATATTCTCCCTTTAGTGAGGGTTAATT |
| GAAA(n) | GAAAGAAAGAAAGAAAGAAAGAAATCTCCCTTTAGTGAGGGTTAATT |
| CAGAA(n) | CAGAACAGAACAGAACAGAACAGAATCTCCCTTTAGTGAGGGTTAATT |
| AAGGAG(n) | AAGGAGAAGGAGAAGGAGAAGGAGAAGGAGTCTCCCTTTAGTGAGGGTTAATT |
| AAGAGG(n) | AAGAGGAAGAGGAAGAGGAAGAGGAAGAGGTCTCCCTTTAGTGAGGGTTAATT |
| AATAC(n) | AATACAATACAATACAATACAATACAATACTCTCCCTTTAGTGAGGGTTAATT |
| AATAG(n) | AATAGAATAGAATAGAATAGAATAGAATAGTCTCCCTTTAGTGAGGGTTAATT |
| AATAGAC(n) | AATAGACAATAGACAATAGACAATAGACTCTCCCTTTAGTGAGGGTTAATT |
| AATAACATAG(n) | AATAACATAGAATAACATAGAATAACATAGTCTCCCTTTAGTGAGGGTTAATT |
| AACAC(n) | AACACAACACAACACAACACAACACAACACTCTCCCTTTAGTGAGGGTTAATT |
| dodeca(n) | ACCGAGTACGGGACCGAGTACGGGTCTCCCTTTAGTGAGGGTTAATT |
| GTGTT(n) | GTGTTGTGTTGTGTTGTGTTGTGTTGTGTTTCTCCCTTTAGTGAGGGTTAATT |
| GTAAT(n) | GTAATGTAATGTAATGTAATGTAATGTAATTCTCCCTTTAGTGAGGGTTAATT |
| GTATT(n) | GTATTGTATTGTATTGTATTGTATTGTATTTCTCCCTTTAGTGAGGGTTAATT |
| TTAA (n) | TTAATTAATTAATTAATTAATTAATTAATTAATCTCCCTTTAGTGAGGGTTAATT |
| CAAT (n) | CAATCAATCAATCAATCAATCAATCAATCAATTCTCCCTTTAGTGAGGGTTAATT |
| AAGAG(n) | GAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAATCTCCCTTTAGTGAGGGTTAATT |
| CTCTT(n) | CTCTTCTCTTCTCTTCTCTTCTCTTCTCTTTCTCCCTTTAGTGAGGGTTAATT |
| 359 Forward | AATTAACCCTCACTAAAGGGAGAAATGGAAATTAAATTTTTTGG |
| 359 Reverse | TTAATACGACTCACTATAGGGAGAGTTTTGAGCAGCTAATTACC |
| chr2R:1,825,641–1825699 sense | GGCAGTTTATGTGCGTACAACAACAACAGGACTGCAAACAAAACACGAAACAGATATTTTTCTCCCTTTAGTGAGGGTTAATT |
| chr2R:1,825,641–1825699 anti-sense | AAAATATCTGTTTCGTGTTTTGTTTGCAGTCCTGTTGTTGTTGTACGCACATAAACTGCCTCTCCCTTTAGTGAGGGTTAATT |
| chr2R:1,826,691–1,826,740 sense | TAGACACATCTACGAAGACACAATTCTACAAGAACTAAACAACAAAAAGTTCTCCCTTTAGTGAGGGTTAATT |
| chr2R:1,826,691–1,826,740 anti-sense | ACTTTTTGTTGTTTAGTTCTTGTAGAATTGTGTCTTCGTAGATGTGTCTATCTCCCTTTAGTGAGGGTTAATT |
| chrX:11,830,844–11,830,910 sense | CCAAGCTTCAGGAGAAAGAGAAAGAAGAAAGCTTTAAACTTAAGGAAAGAGAAGAGAGCCTTAGGATTCTCCCTTTAGTGAGGGTTAATT |
| chrX:11,830,844–11,830,910 antisense | CTAAGGCTCTCTTCTCTTTCCTTAAGTTTAAAGCTTTCTTCTTTCTCTTTCTCCTGAAGCTTGGCTTTCTCCCTTTAGTGAGGGTTAATT |
| chrX:12,660,096–12,660,145 sense | TCGCACACACACACGCAACACTTAGGCACACATAGGAGATAGAGTGAGATCTCCCTTTAGTGAGGGTTAATT |
| chrX:12,660,096–12,660,145 anti-sense | TCTCACTCTATCTCCTATGTGTGCCTAAGTGTTGCGTGTGTGTGTGCGATCTCCCTTTAGTGAGGGTTAA TT |
| chrX:22,453,019–22,453,076 sense | CGACAGACAGTAAAATTAAACAAACTGCGGACGCGTGTGACAGAACTAATCCAACTTTCTCCCTTTAGTGAGGGTTAATT |
| chrX:22,453,019–22,453,076 anti-sense | AAGTTGGATTAGTTCTGTCACACGCGTCCGCAGTTTGTTTAATTTTACTGTCTGTCGTCTCCCTTTAGTGAGGGTTAATT |
| chr3R:3,169,758–3,169,820 antisense | TCGGAAGAGACTAAACTTGTGCATTCGATATAGCTCTTTGTCGGCCCTAGCTGCTGTAAACAATCTCCCTTTAGTGAGGGTTAATT |
| chr3R:3,169,758–3,169,820 sense | TTGTTTACAGCAGCTAGGGCCGACAAAGAGCTATATCGAATGCACAAGTTTAGTCTCTTCCGATCTCCCTTTAGTGAGGGTTAATT |
| chr3R:3,170,372–3,170,441 antisense | TTAAACTATATTAAACATTGTATATAAGTATAATAGCGAATACTATTTACGTATATGTTCT TTCATAAATTCTCCCTTTAGTGAGGGTTAATT |
| chr3R:3,170,372–3,170,441 sense | ATTTATGAAAGAACATATACGTAAATAGTATTCGCTATTATACTTATATACAATGTTTAATATAGTTTAATCTCCCTTTAGTGAGGGTTAATT |
Table 3
Antibodies used for Immuno-Fluorescence.
| Antibody | Supplier; Cat. number | Working concentration |
|---|---|---|
| Rabbit-anti H3K9me3 | Abcam; 8898 | 1/250 |
| Mouse-anti H3K9me2 | Active Motif; 39753 | 1/250 |
| Rabbit-anti-H2AV | Lake placid AM318; 9751 | 1/100 |
| Goat anti-GFP | Rockland 121600-101-215 | 1/500 |
| Rabbit anti-H4acetyl | Millipore 06–598 | 1/200 |
| Rat anti-Mst77F | Elaine Dunleavy, PhD; NUI Galway, Ireland | 1/200 |
| Guinea pig anti-Mst35Ba/Bb (Protamine A/B) | Elaine Dunleavy, PhD; NUI Galway, Ireland | 1/200 |
| Mouse anti pan-histone | Millipore MAB 3422 | 1/200 |
Table 4
Uniquely mapped RNA identified via phrap adjacent AAGAG(>10) containing blocks
| Chr | e0-2hr | e2-4hr | e4-8hr | e8-12hr | e12-14hr | e14-16hr | e16-20hr | e20-24hr |
|---|---|---|---|---|---|---|---|---|
| 2R | NA | NA | NA | NA | NA | NA | chr2R.1825640.1825699 | NA |
| X | NA | NA | NA | NA | NA | NA | NA | chrX.12660077.12660134 |
| X | NA | NA | NA | NA | NA | NA | chrX.11830795.11830858 | NA |
| X | chrX.22453019.22453120 | NA | chrX.22453019.22453182 | NA | chrX.22453019.22453163 | chrX.22453019.22453177 | chrX.22453019.22453093 | chrX. 22453019.22453196 |
Table 5
shRNA and overexpression oligos.
| Description | Sequence 5’−3’ |
|---|---|
| shRNA to AAGAG(n) | ctagcagtGAAGAGAAGAGAAGAGAAGAGtagttatattcaagcataCTCTTCTCTTCTCTTCTCTTCgcg |
| shRNA to AAGAG(n) complement | aattcgcGAAGAGAAGAGAAGAGAAGAGtatgcttgaatataactaCTCTTCTCTTCTCTTCTCTTCactg |
| shRNA to scrambled | ctagcagtGAGAGAAAAAGGGAAAGAAGGtagttatattcaagcataCCTTCTTTCCCTTTTTCTCTCgcg |
| shRNA to scrambled complement | aattcgcGAGAGAAAAAGGGAAAGAAGGtatgcttgaatataactaCCTTCTTTCCCTTTTTCTCTCactg |
| AAGAG(37) for over-expression | ATCAAGACTGCTAGCAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAGAAGAG |
| AAGAG(37) over-expression complement | CCATTGACTGAATTCCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTTCTCTT |
Table 6
Fly lines.
| Stock name or genotype | Obtained from: stock number | Description | |
|---|---|---|---|
| y[1] v[1]; P{y[+t7.7]=CaryP}attP40 | Bloomington: 36304 | Background strain for insertion of pValium20 vector containing shRNA | |
| y[1] v[1]; P{y[+t7.7]=CaryP}attP2 | Bloomington: 36303 | Background strain for insertion of pValium vector containing AAGAG expression construct. | |
| y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20-mCherry}attP2 | Bloomington: 35785 | Control strain for RNAi. Expresses dsRNA to mCherry | |
| y[1] v[1]:UAS-AAGAG shRNA:: | Rainbow Transgenic Flies, Inc | Expresses shRNA under UAS promoter targeting AAGAG(n) | |
| y[1] v[1]:UAS-scramble shRNA:: | Rainbow Transgenic Flies, Inc | Expresses shRNA under UAS promoter targeting random AG containing sequences | |
| y[1] w[67c23]; P{w[+mC]=dpp-GAL4.PS}6A/TM3, Ser[1] | Bloomington: 7007 | Dpp-GAL4 | |
| y[1] v[1]::UAS-AAGAG(37) | Rainbow Transgenic Flies, Inc | Expresses a 187 base repeat of AAGAG RNA under a UAS promoter | |
| C(1;Y)1, y[1] w[A738]: y[+]/0 and C(1)RM, y[1] v[1]/0 | Bloomington:2494 | XO (Y chromosome deficient males) | |
| y[*] w[*]; P{w[+mW.hs]=GawB}NP1233/CyO, P{w[-]=UAS lacZ.UW14}UW14 | Kyoto: 103948 | Fascillin-GAL4 | |
| y[*] w[*]; P{w[+mW.hs]=GawB}NP1624/CyO, P{w[-]=UAS lacZ.UW14}UW14 | Kyoto:104055 | Traffic Jam-GAL4 | |
| w[*]; P{w[+mW.hs]=GawB}ptc[559.1] | Kyoto: 103948 | PTC-GAL4 | |
| :: nanos-Gal4, dcr2-UAS/TM3 sb | Unknown | Nanos-GAL4 | |
| w;;bamGAL4, UAS-dicer2 | Unknown | Bam-GAL4 | |
| y[1] w[*]::P{w[+mC]=Act5 C-GAL4}17bFO1/TM6B, Tb[1] | Bloomington: stock 3954 | Expresses GAL4 ubiquitously under control of Act5C promoter |
Table 7
Quantification of post-canoe stage sperm DNA morphological defects in 4–7 day old testes.
| N | Normal bundle | Lagging bundle | Kinked | Knotted | Needle eyed | Decondensed | |
|---|---|---|---|---|---|---|---|
| Scrambled RNAi | 1 | 2 | 2 | 4 | 0 | 0 | 0 |
| 2 | 9 | 7 | 6 | 0 | 0 | 0 | |
| 3 | 21 | 5 | 0 | 0 | 0 | 0 | |
| 4 | 5 | 1 | 0 | 0 | 0 | 0 | |
| 5 | 29 | 1 | 0 | 0 | 0 | 0 | |
| 6 | 6 | 2 | 0 | 0 | 0 | 0 | |
| AAGAG RNAi | 1 | 0 | 8 | 2 | 0 | 0 | 2 |
| 2 | 1 | 8 | 0 | 2 | 2 | 0 | |
| 3 | 0 | 1 | 5 | 2 | 1 | 0 | |
| 4 | 0 | 0 | 1 | 0 | 0 | 0 | |
| 5 | 0 | 1 | 2 | 1 | 1 | 0 | |
| 6 | 0 | 1 | 5 | 1 | 0 | 0 | |
| 7 | 0 | 0 | 3 | 3 | 0 | 0 | |
| 8 | 0 | 0 | 0 | 0 | 1 | 0 | |
| 9 | 1 | 2 | 4 | 1 | 1 | 0 | |
| 10 | 3 | 5 | 2 | 4 | 2 | 0 | |
| 11 | 2 | 2 | 14 | 0 | 1 | 0 | |
| 12 | 1 | 4 | 9 | 1 | 1 | 0 | |
| AAGAG RNA (Rescue) | 1 | 0 | 1 | 2 | 0 | 0 | 0 |
| 2 | 5 | 2 | 2 | 0 | 0 | 0 | |
| 3 | 7 | 1 | 1 | 0 | 0 | 0 | |
| 4 | 8 | 3 | 6 | 3 | 0 | 0 | |
| 5 | 0 | 0 | 9 | 0 | 0 | 0 | |
| 6 | 4 | 4 | 6 | 0 | 0 | 0 | |
| 7 | 8 | 4 | 3 | 0 | 0 | 0 | |
| 8 | 9 | 6 | 7 | 0 | 0 | 0 | |
| 9 | 11 | 1 | 5 | 0 | 0 | 0 | |
| 10 | 8 | 2 | 4 | 0 | 0 | 0 | |
| 11 | 3 | 0 | 2 | 0 | 0 | 0 | |
| 12 | 3 | 0 | 6 | 1 | 0 | 0 | |
| 13 | 2 | 2 | 8 | 0 | 0 | 0 | |
| 14 | 5 | 7 | 5 | 0 | 1 | 0 | |
| 15 | 5 | 0 | 3 | 0 | 0 | 0 | |
| 16 | 5 | 5 | 5 | 1 | 0 | 0 | |
| 17 | 3 | 4 | 3 | 0 | 0 | 0 | |
| 18 | 5 | 1 | 6 | 0 | 0 | 0 | |
| 19 | 3 | 11 | 5 | 1 | 0 | 0 | |
| 20 | 3 | 2 | 1 | 0 | 1 | 0 |
Table 8
Quantification of canoe stage DNA morphological defects, in 4–7 day old testes
| N | Normal canoe | Abnormal canoe | |
|---|---|---|---|
| Scrambled RNAi | 1 | 2 | 0 |
| 2 | 7 | 1 | |
| 3 | 6 | 1 | |
| 4 | 3 | 5 | |
| 5 | 9 | 1 | |
| 6 | 5 | 1 | |
| AAGAG RNAi | 1 | 1 | 0 |
| 2 | 0 | 1 | |
| 3 | 0 | 1 | |
| 4 | 1 | 1 | |
| 5 | 0 | 0 | |
| 6 | 0 | 1 | |
| 7 | 0 | 0 | |
| 8 | 2 | 4 | |
| 9 | 1 | 2 | |
| 10 | 0 | 2 | |
| 11 | 1 | 2 | |
| 12 | 3 | 6 | |
| 13 | 2 | 7 | |
| 14 | 1 | 5 | |
| AAGAG RNA (Rescue) | 1 | 7 | 4 |
| 2 | 3 | 2 | |
| 3 | 0 | 3 | |
| 4 | 0 | 4 | |
| 5 | 0 | 3 | |
| 6 | 0 | 0 | |
| 7 | 1 | 4 | |
| 8 | 3 | 4 | |
| 9 | 6 | 8 | |
| 10 | 3 | 3 | |
| 11 | 1 | 0 | |
| 12 | 2 | 2 | |
| 13 | 8 | 9 | |
| 14 | 1 | 6 | |
| 15 | 0 | 2 | |
| 16 | 7 | 9 | |
| 17 | 0 | 1 | |
| 18 | 3 | 2 | |
| 19 | 2 | 4 | |
| 20 | 1 | 1 |
Table 9
qPCR oligos.
| mRNA target | Sequence 5’−3’ |
|---|---|
| Actin-5c Forward | CAGCCAGCAGTCGTCTAATC |
| Actin-5c Reverse | ACAACCAGAGCAGCAACTTC |
| Rpl32 Forward | CGATGTTGG GCATCAGATAC |
| Rpl32 Reverse | CCCAAGATCGTGAAGAAGC |
| pip5K59B Forward | CTCCTGCTCTGCTATCGTATTC |
| pip5K59B Reverse | AGAGGAGCCATCAACATCAC |
| Peb Forward | TGGTTGGACCGCTTAACATAG |
| Peb Reverse | GCGACACCAAGAGCCATAA |
| CG33080 Forward | ATTACGATCGCGGGCTTATC |
| CG33080 Reverse | CGGTTCTAGGAGCACTGATATAAA |
