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. 2021 Apr 13;40(12):e105763. doi: 10.15252/embj.2020105763
REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Chicken anti GFP 1:500 AVES

GFP‐1020

RRID: AB_10000240

Mouse monoclonal anti FasII 1:25 Developmental Studies Hybridoma Bank (DSHB)

1D4

RRID: AB_528235

Mouse monoclonal anti Brp 1:5 DSHB

nc82

RRID: AB_2314866

Rabbit anti TH 1:500 Merck Millipore

AB152

RRID: AB_390204

Rat anti RFP 1:500 ChromoTek

5f8

RRID: AB_2336064

Alexa fluor 568 Goat anti Rat 1:500 Invitrogen

A‐21247

RRID: AB_2534121

Alexa fluor 647 Goat anti Mouse 1:500 Invitrogen

A‐32728

RRID: AB_2633277

Alexa fluor 488 Goat anti Mouse 1:500 Invitrogen

A‐11001

RRID: AB_2534069

FITC Goat anti Chicken 1:500 Invitrogen

A‐16055

RRID: AB_2534728

Alexa fluor 568 Goat anti Rabbit 1:500 Invitrogen

A‐11036

RRID: AB_10563566

Bacterial and Virus Strains
DH5ɑ
Chemicals, Peptides, and Recombinant Proteins
Cell Dissociation Solution Sigma Aldrich

Cat# C1544
Collagenase/Dispase mix Roche Cat# 10269638001
poly‐L‐lysine Sigma Aldrich Cat# P1524‐25MG
Critical Commercial Assays
Pico pure RNA isolation kit Thermo Fisher Cat# KIT0204
Gibson assembly NEB Cat# E5510S
Deposited Data
Raw data files for UNF Perturbation seq Deposited in the NCBI Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE165896)
Experimental Models: D. melanogaster

w[1118]; P{y[+t7.7] w[+mC]=GMR71G10‐GAL4}attP2

(R71G10‐Gal4)

 Bloomington Drosophila Stock Center (BDSC)

BDSC: 39604

FlyBase ID (FBID):

FBsf0000166728
y*,w*; P{y[+t7.7] w[+mC]=GMR71G10‐GAL4}attP40 (Alyagor et al, 2018) N/A
P{GawB}elavC155 (C155‐GAL4) BDSC

BDSC: 458

FBID: FBti0002575
w1118; P{GAL4}repo/TM3, Sb1 (Repo‐Gal4) BDSC

BDSC: 7415

FBID: FBti0018692
w[1118]; P{y[+t7.7] w[+mC]=GMR58E02‐GAL4}attP2 (R58E02‐Gal4) BDSC

BDSC: 41347

FBID: FBtp0061564
w[1118]; P{y[+t7.7] w[+mC]=GMR18H09‐GAL4}attP2 (R18H09‐Gal4) BDSC

BDSC: 48830

FBID: FBti0133650
w[1118]; P{y[+t7.7] w[+mC]=GMR48H11‐GAL4}attP2 (R48H11‐GAL4) BDSC

BDSC: 50396

FBID: FBti0136291
w[1118]; P{y[+t7.7] w[+mC]=GMR10G03‐GAL4}attP2 (R10G03‐Gal4) BDSC

BDSC: 48271

FBID: FBti0132904
P{y[+t7.7] w[+mC]= GMR71G10‐QF2HSP}attP40 (R71G10‐QF2) This study N/A
y[1] w[*]; PBac{y[+mDint2] w[+mC]=10XQUAS‐6XGFP}VK00018, P{w[+mC]=UAS‐mtdTomato‐3xHA}2; P{y[+t7.7] w[+mC]=GMR58E02‐QF2.L}attP2 (R58E02‐QF2) BDSC

BDSC: 66480

FBID: FBti0184753
w[1118]; P{y[+t7.7] w[+mC]=R53C03‐p65.AD}attP40; P{y[+t7.7] w[+mC]=R24E12‐GAL4.DBD}attP2/TM6B, Tb[1] (MB298B‐Gal4) BDSC

BDSC: 68309

FBID: FBst0068309
w[1118]; P{y[+t7.7] w[+mC]=R30G08‐p65.AD}attP40; P{y[+t7.7] w[+mC]=R48B03‐GAL4.DBD}attP2 (MB441B‐Gal4) BDSC

BDSC: 68251

FBID: FBst0068251
w[*]; P{w[+mW.hs]=GawB}OK107 ey[OK107] (OK107‐Gal4) BDSC

BDSC: 854

FBID: FBti0004170

w[1118]; P{y[+t7.7] w[+mC]=R94B10‐GAL4.DBD}attP2 PBac{y[+mDint2] w[+mC]=R52G04‐p65.AD}VK00027

(MB083C‐Gal4)

 BDSC

BDSC: 68287

FBID: FBst0068287
y*,w*; DIP‐δ‐T2A‐Gal4 This study N/A

w[*]; P{y[+t7.7] w[+mC]=10XUAS‐IVS‐mCD8::GFP}attP2

(UAS‐CD8‐GFP)

 BDSC

BDSC: 32185

FBID: FBst0032185
w[*]; P{y[+t7.7] w[+mC]=10XUAS‐IVS‐mCD8::GFP} attP40 (UAS‐CD8‐GFP) BDSC

BDSC: 32186

FBID: FBst0032185
y[1] w[*] P{y[+t7.7] w[+mC]=10XUAS‐IVS‐mCD8::GFP}su(Hw)attP8 (UAS‐CD8‐GFP) BDSC

BDSC: 32189

FBID: FBst0032189:

y[1] w[*]; P{w[+mC]=UAS‐CD4‐tdTom}7M1

(UAS‐CD4‐tdT)

 BDSC

BDSC: 35841

FBID: FBst0035841

y[1] w[1118]; P{w[+mC]=QUAS‐mtdTomato‐3xHA}26

(QUAS‐mtdT)

 BDSC

BDSC: 30005

FBID: FBti0129951
w[1118]; P{w[+mC]=UAS‐Dcr‐2.D}10 BDSC

BDSC: 24651

FBID: FBst0024651

P{hsFLP}22, w*, P{w[+mC]=UAS‐mCD8::GFP.L}; P{w[+mC]=tubP‐GAL80}LL10 P{ry[+t7.2]=neoFRT}40A; GMR71G10‐Gal4, P{w[+mC]=UAS‐Dcr‐2.D}10

(recombination between R71G10‐Gal4 and UAS‐Dcr‐2)

 This study

N/A

FBIC: FBrf0227179
P{hsFLP}22, w*, P{w[+mC]=UAS‐mCD8::GFP.L}; P{w[+mC]=tubP‐GAL80}LL10 P{ry[+t7.2]=neoFRT}40A; DIP‐δ‐T2A‐Gal4 This study N/A
y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF03306}attP2 (RNAi of Dpr5) BDSC

BDSC: 29627

FBID: FBst0029627
y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF03172}attP2 (RNAi of Dpr8) BDSC

BDSC: 28744

FBID: FBst0028744
y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS00288}attP2 (RNAi of Dpr9) BDSC

BDSC: 33409

FBID: FBst0033409
y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02920}attP2 (RNAi of Dpr10) BDSC

BDSC: 27991

FBID: FBst0027991
y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF03210}attP2 (RNAi of Dpr12) BDSC

BDSC: 28782

FBID: FBst0028782
[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.GL01238}attP2 (RNAi of Dpr17) BDSC

BDSC: 41656

FBID: FBst0041656
y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF03283}attP2 (RNAi of Dpr18) BDSC

BDSC: 29604

FBID: FBst0029604
[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02923}attP2 (RNAi of Dpr20) BDSC

BDSC: 28293

FBID: FBst0028293
P{ry[+t7.2]=hsFLP}12, y[1] w[*]; P{y[+t7.7] w[+mC]=UAS‐Cas9.P2}attP40 BDSC

BDSC: 58985

FBID: FBst0058985
w[1118]; P{y[+t7.7] w[+mC]=UAS‐Cas9.C}attP2 BDSC

BDSC: 54595

FBID:FBst0054595
gRNA dpr12 This study N/A
y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TKO.GS02451}attP40 (gRNA DIP‐δ) BDSC

BDSC: 78754

FBID: FBst0078754
y*,w*;FRT40A, dpr12∆50‐81, FRTG13, cn,bw This study N/A
w*; DIPδ1‐119, FRT2A A generous gift from Larry Zipursky, UCLA N/A
y[1] w[67c23]; Mi{PT‐GFSTF.1}dpr12[MI01695‐GFSTF.1]/SM6a (dpr12GFSTF) BDSC

BDSC: 60171

FBID: FBst0060171
y[1] w[*]; Mi{PT‐GFSTF.1}DIP‐delta[MI08287‐GFSTF.1] (DIP‐δGFSTF) BDSC

BDSC: 60558

FBID: FBst0060558
y[1] w[*]; Mi{y[+mDint2]=MIC}DIP‐delta[MI08287] BDSC

BDSC: 51229

FBID: FBst0051229
y[1] w[*]; P{w[+mC]=lox(Trojan‐GAL4)x3}10; Dr[1]/TM3, Sb[1] Ser[1] BDSC

BDSC: 60310

FBID: FBst0060310
P{y[+mDint2]=Crey}1b, y[1] M{vas‐int. Dm}ZH‐2A w[*] BDSC

BDSC: 60299

FBID: FBst0060299
w[*]; P{w[+mC]=UAS‐Cbbeta\DT‐A.I}18/CyO (UAS‐DTI) BDSC

BDSC: 25039

FBID: FBst0025039
UAS‐Dpr12 (in86FB) This study N/A
UAS‐DIP‐δ (in attp40) This study N/A
UAS‐DIP‐δ‐T2A‐tdT A generous gift from Larry Zipursky, UCLA N/A
UAS‐DIP‐α A generous gift from Larry Zipursky, UCLA N/A
UAS‐DIP‐α‐T2A‐tdT A generous gift from Larry Zipursky, UCLA N/A
UAS‐FasII‐A A generous gift form Brian McCabe (Beck et al, 2012) N/A
y[1] M{w[+mC]=nos‐Cas9.P}ZH‐2A w[*] BDSC

BDSC: 54591

FBID: FBst0054591
UAS‐DIP‐δ‐RNAi (in attp40) This study N/A
y[1] w[*] P{y[+t7.7] w[+mC]=10XUAS‐IVS‐mCD8::RFP}su(Hw)attP8 BDSC

BDSC: 32220

FBID: FBst0032220
Oligonucleotides

gRNAs for Dpr12 CRISPR deletion:

CGCAGTTCCATCAGGTGCAGGGG

CCATTAGACATATCTTCCTGACC

 This study N/A

Primers for dpr12∆50‐81 check PCR:

F’:GTTGCCGTAGCTGAAAGGATT

R’:TAAACCGGGTATCGGAGTGTC

 This study N/A

RNAi for DIPδ

(SS) GACGAUAAGAAACCUACAAUA

(AS) UUGUAGGUUUCUUAUCGUCAG

 This study N/A

Primers for QF2 cloning

F’:TAAGCCAACTTTGAATCACAAGACGCATACCAAACGGTACATGCCACCCAAG

R’:TGAATAATTTTCTATTTGGCTTTAGTCGACGGTATCGATAATCACTGTTCGT

 This study N/A

Primers for hsp70 cloning

F’:AAGTGGTGATAAACGGCCGGCCGAGCGCCGGAGTATAAATAGAG

R’:AAGTGGTGATAAACGGCCGGCCGAGCGCCGGAGTATAAATAGAG

 This study N/A
Recombinant DNA
pCFD4 (Port et al, 2014) N/A
pVALIUM22 Harvard Medical School PlasmID: pVALIUM22
pDEST‐UAS‐IVS‐Syn21‐p10aw (Rabinovich et al, 2016) N/A
pBPGUw addgene Plasmid #17575
pBPGUw‐QF2 This study N/A
GMR71G10 entry vector (Alyagor et al, 2018) N/A
GMR71G10‐QF2hsp70 This study N/A
Plasmid: UAS‐IVS‐Syn21‐Dpr12‐p10 This study N/A
Plasmid: UAS‐IVS‐Syn21‐DIP‐δ‐p10 This study N/A
Plasmid: pCFD4‐dpr12 (Dpr12 gRNA) This study N/A
Plasmid: pVALIUM22‐DIP‐δ (DIP‐δ RNAi) This study N/A
Software and Algorithms
FIJI Image J https://imagej.net/Fiji/Downloads
VVD Viewer (a branch of FluoRender, Center for Integrative Biomedical Computing (CIBC), Utah) Takashi Kawase https://github.com/takashi310/VVD_Viewer/releases
MATLAB R2016a software MathWorks N/A
FlyMine (Lyne et al, 2007) http://www.flymine.org/
HISAT v.0.1.5 (Kim et al, 2015) https://github.com/infphilo/hisat
DEseq2 (Love et al, 2014) N/A
Gene‐e v.3.0.215 Broad Institute, Inc. https://software.broadinstitute.org/GENE‐E/
HOMER software (Heinz et al, 2010)
DSIR (Vert et al, 2006) http://biodev.extra.cea.fr/DSIR/DSIR.php
FlyCRISPR (Gratz et al, 2014) http://flycrispr.molbio.wisc.edu/
neuPrint (Clements et al, 2020) https://neuprint.janelia.org/
Other
Zeiss LSM 800 confocal microscope Zeiss
40× 1.3 NA oil immersion lens Zeiss