Coordinated repression of pro-differentiation genes via P-bodies and transcription maintains Drosophila intestinal stem cell identity

. Author manuscript; available in PMC: 2023 Jan 24.

Published in final edited form as: Curr Biol. 2021 Dec 6;32(2):386–397.e6. doi: 10.1016/j.cub.2021.11.032

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Mouse anti-V5	Bio-Rad	Cat# MCA1360GA; RRID:AB_567249
Mouse anti-FLAG	Sigma-Aldrich	Cat# F3165; RRID:AB_259529
Mouse anti-FMRP	DSHB	Cat# 5A11; RRID:AB_528252
Mouse anti-Prospero	DSHB	Cat# MR1A; RRID:AB_528440
Mouse anti-Delta	DSHB	Cat# C594.9B; RRID:AB_528194
Mouse anti-Me31B	³⁴	N/A
Mouse anti-Tubulin	DSHB	Cat# 12G10; RRID:AB_1157911
Rabbit anti-EDC3	This study	N/A
Rabbit anti-GFP	Thermo Fisher Scientific	Cat# A11122; RRID:AB_221569
Rabbit anti-HA	Cell Signaling Technology	Cat# 3724S; RRID:AB_1549585
Rabbit anti-Me31B	³²	N/A
Rabbit anti-GE1	³³	N/A
Rabbit anti-Pdm1	A gift from Xiaohang Yang	N/A
Rabbit anti-PATR-1	⁴⁵	N/A
Rat anti-TRAL	This study	N/A
Rat anti-EDC3	This study	N/A

Chemicals, peptides, and recombinant proteins
Paraformaldehyde solution	Electron Microscopy Sciences	Cat# 15714
Bleomycin	Calbiochem	Cat# 203408
TRIzol^® LS reagent	Ambion	Cat# 10296028
Elastase	Sigma-Aldrich	Cat# E0258
10X PBS pH 7.4, RNase-free	Invitrogen	Cat# AM9624
Heptane	Sigma-Aldrich	Cat# 246654
Triton X-100	Sigma-Aldrich	Cat# 11332481001
Vectashield	Vector Laboratories	Cat# H-1000
ProLong Diamond Antifade	Invitrogen	Cat# P36965
Krebs-Ringer media	Alfa Aesar	Cat# J67591
Rapamycin	LC Laboratories	Cat# r-5000
ECL-detection reagent 1 and 2	Thermo Fisher Scientific	Cat# 1859701
SUPERasIn RNase Inhibitor	Invitrogen	Cat# AM2696
cOmplete Protease Inhibitor	Roche	Cat# 1183617001
Turbo DNase	Thermo Fisher Scientific	Cat# AM2239
Superscript III	Thermo Fisher Scientific	Cat# 56575
PowerUp SYBR Green Master Mix	Thermo Fisher Scientific	Cat# A25742

Critical commercial assays
RNAscope Multiplex Fluorescent Reagent Kit v2	ACD	Cat# 323100
Ovation® SoLo RNA-seq system	Tecan	Cat# 0502 including 0407
ApopTag® Fluorescein In situ Apoptosis Detection Kit	Sigma-Aldrich	Cat# S7110

Deposited data
Patr-1 RNA-seq data: GSE183996	This paper	GSE183996
esg RNA-seq data: ArrayExpress E-MTAB-2915	⁵³	N/A
vtd RNA-seq data: Provided by H. Jasper	⁵²	N/A

Experimental models: Organisms/strains
D. melanogaster: Patr-1^P105FS1 / TM3, Sb[1]	This study	N/A
D. melanogaster: Patr-1^P105FS2 / TM3, Sb[1]	This study	N/A
D. melanogaster: Patr-1^P106FS1 / TM3, Sb[1]	This study	N/A
D. melanogaster: Patr-1^P107FS1 / TM3, Sb[1]	This study	N/A
D. melanogaster: Patr-1^P104Δ / TM3, Sb[1]	This study	N/A
D. melanogaster: Patr-1^P105Δ / TM3, Sb[1]	This study	N/A
D. melanogaster: P21M20	⁴⁵	N/A
D. melanogaster: esg-Gal4 UAS-GFP tubGal80 ^ts	³⁰	N/A
D. melanogaster: gbe-smGFP::V5::nls	⁴⁸	N/A
D. melanogaster: hsFLP12 , tubGAL4, UAS-GFP / + ; P{lin-28 13kb::mCherry}attP40 / Cyo ; FRT82B tubP-GAL80LL3 / TM6, Tb[1]	This study	N/A
D. melanogaster: {3Xgbe-GAL80}ZH-2A ; {mira-KDRT>-dSTOP-KDRT>-GAL41438}attP40, P{tubP-GAL80[ts]}20; {CG10116-KD.PEST1107}attP2	⁴⁸	N/A
D. melanogaster: w; Su(h)Gbe-Gal4, UAS-mCD8GFP/ CyO; tubGal80^ts / TM6B	B. Edgar	N/A
D. melanogaster: rin::HA	¹⁸	N/A
D. melanogaster: P{w[+mC]=UAS-rpr.C}27	BDSC	RRID:BDSC_5823
D. melanogaster: y[1] w[67c23]; P{y[+mDint2] w[+mC]=EPgy2}Patr-1[EY10289] / TM3, Sb[1] Ser[1]	BDSC	RRID:BDSC_19805
D. melanogaster: P{y[+t7.7] v[+t1.8]=TRiP.HM05052}attP2	BDSC	RRID:BDSC_28566
D. melanogaster: P{y[+t7.7] v[+t1.8]=TRiP.HM05072}attP2	BDSC	RRID:BDSC_28584
D. melanogaster: P{y[+t7.7] v[+t1.8]=TRiP.JF03061}attP2	BDSC	RRID:BDSC_28646
D. melanogaster: P{y[+t7.7] v[+t1.8]=TRiP.HMS00857}attP2	BDSC	RRID:BDSC_33914
D. melanogaster: P{y[+t7.7] v[+t1.8]=TRiP.HMS01144}attP2 / TM3, Sb[1]	BDSC	RRID:BDSC_34667
D. melanogaster: P{y[+t7.7] v[+t1.8]=TRiP.GL00680}attP2	BDSC	RRID:BDSC_38908
D. melanogaster: P{y[+t7.7] v[+t1.8]=TRiP.HMJ22374}attP40	BDSC	RRID:BDSC_58288
D. melanogaster: P{w+mC=UAS-Stinger}2	BDSC	RRID:BDSC_65402
D. melanogaster: P{UAS-nub.ORF.3xHA.GW}attP2	BDSC	RRID:BDSC_83303
D. melanogaster: y¹ sc v¹ sev²¹; P{nos-Cas9}attP2*	BDSC	RRID:BDSC_78782
D. melanogaster: P{EPgy2}Patr-1 ^EY10289	BDSC	RRID:BDSC_19805
D. melanogaster: M{UAS-esg.ORF.3xHA.GW}ZH-86Fb	FlyORF	RRID:FlyBase_FBst0501340

Oligonucleotides
nub qPCR forward primer: 5'-TGGTGGGGCATTGAATTTAACC-3'	This study	N/A
nub qPCR reverse primer: 5'-CTGTGGCCGATTTCACCGAAT-3'	This study	N/A
Patr-1 qPCR forward primer: 5'-TCGTTTTTCGGCTTTGACACG-3'	This study	N/A
Patr-1 qPCR reverse primer: 5'-GTCATTGAGGGCATCGTATTCC-3'	This study	N/A
esg qPCR forward primer: 5'-CTGAGATCCCAGCCATGTT-3'	This study	N/A
esg qPCR reverse primer: 5'-CTGCTTGAAGTTGTGTGTCTG-3'	This study	N/A
Tsp2A qPCR forward primer: 5'-CGTCTGGTTGAGGGCTGAG-3'	This study	N/A
Tsp2A qPCR reverse primer: 5'-CACGTACACGCCGATGTAG-3'	This study	N/A
MRP qPCR forward primer: 5'-AATCGAAAGTATGGCGTGCAG-3'	This study	N/A
MRP qPCR reverse primer: 5'-GGGGAATCGACAGCACAGT-3'	This study	N/A
bbg qPCR forward primer: 5'-AGGATGAGTCCTCTGCACCA-3'	This study	N/A
bbg qPCR reverse primer: 5'-TCCTGGCGATATACTTTCCTGG-3'	This study	N/A
gRNA #1: 5'-CATGTTGAACATGTTATACATGG-3'	This study	N/A
gRNA #2: 5'-TGTGACGAGACTGTCGGAAGGGG-3'	This study	N/A
EDC3 amplification forward primer: 5'-GGGAATTCATGGGTCCGACGGATCAAGA-3'	This study	N/A
EDC3 amplification reverse primer: 5'-GGGGCGGCCGCTCACTTATCGGCACTTATCTCGA-3'	This study	N/A

Software and algorithms
ImageJ/Fiji	³⁷	https://imagej.net/; RRID:SCR_003070
Leica LAS-X	Leica	https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/; RRID:SCR_013673
Prism Version 9.0	GraphPad Software	http://www.graphpad.com/; RRID:SCR_002798
RNA-seq analysis pipeline	⁴⁸	https://github.com/jkkbuddika/RNA-Seq-Data-Analyzer

Other
nub in situ probes	ACD	Cat# 523981
Opal 620	AKOYA Biosciences	Cat# FP1495001KT
Chemiluminescence films	GE Healthcare	Cat# 28906839
Immobilon^®-P membrane	Millipore	Cat# IPVH00010
4–20% gradient polyacrylamide gel	Bio-Rad	Cat# 456–1093
Leica SP8 Scanning Confocal microscope	Leica	N/A
DeltaVision OMX system	DeltaVision	N/A