Gasperini et al Supplementary Material

This file includes: • Supplementary Material • Supplementary Figures S1-S3 • Supplementary data Table S1 (legend) • Supplementary data Table S2 (legend)

List of antibodies used for WB:

Antibody Host Company Catalog Dilution

Mouse Santa Cruz sc-377347 1:100 MILI Rabbit Kind gift of Dr. Hannon 1:150

MIWI Rabbit Kind gift of Dr. Hannon 1:200

ACTIN Rabbit Abcam ab13970 1:1000

GADPH Rabbit Santa Cruz sc-25778 1:1000

GFAP Rabbit Dako Z-0334 1:1000

List of antibodies used for IF/IHC:

Antibody Host Company Catalog Dilution

MILI rabbit Hannon Lab 1:100

BrdU rat Abcam ab6326 1:200

KI67 rabbit Abcam ab15580 1:250

GFAP rabbit Dako Z-0334 1:1000

NeuN mouse Millipore MAB377 1:250

RPL26 rabbit Abcam ab59567 1:500

Nestin mouse Millipore MAB353 1:250

Cleaved Caspase-3 rabbit Cell Signaling Technology 9664 1:400

List of Primers used for real time qPCR

Assay ID Forward primer (5’ to 3’) Reverse primer (5’ to 3’)

Actin GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT

Mili GGCCAGCATAAATCTCACAC TAGCTGGCCATCAGACACTC

Miwi TAATTGGCCTGGAGTCATCC GAGGTAGTAGAGGCGGTTGG

Gfap GGGGCAAAAGCACCAAAGAAG GGGACAACTTGTATTGTGAGCC

Complement C3 CCAGCTCCCCATTACGTCTG GCACTTGCCTCTTTAGGAAGTC

Serpina 3n ATTTGTCCCAATGTCTGCGAA TGGCTATCTTGGCTATAAAGGGG

1 Gasperini et al Supplementary Material

Cxcl10 CCAAGTGCTGCCGTCATTTTC GGCTGGCAGGGATGATTTCAA

Cyclin A GCCTTCACCATTCATGTGGAT TTGCTGCGGGTAAAGAGACAG

Cyclin D1 GCGTACCCTGACACCAATCTC CTCCTCTTCGCACTTCTGCTC

Cyclin E GATCCAGAAAAAGGAAGGCAAA TGAAGAAATTGCCAAGATTGACA

Assay ID Forward primer (5’ to 3’) Reverse primer (5’ to 3’) Probe (5’ to 3’)

L1 5’UTR Tf TGAGCACTGAAACTCAGAGGAG GATTGTTCTTCTGGTGATTCTGTT FAM- A GAATCTGTCTCCCAGGTCTG- MGBNFQ SINE B1 TGGCGCACGCCTTTAATC GAGACAGGGTTTCTCTGTGTAGC FAM-CAGAGGCAGGCGGAT- C MGBNFQ 5s rRNA ACGGCCATACCACCCTGAA GGTCTCCCATCCAAGTACTAACC FAM-CCGAGATCAGACGAGAT- A MGBNFQ Ubiquitin C ACAGACGTACCTTCCTCACC CCCCATCACACCCAAGAACA VIC- AAAAAGAGCCCTCCTTGTGC- MGBNFQ

2 Gasperini et al Supplementary Material shMILI DIV0 MIWI

Supplementary figures

A CTL KD CTL KD B C CONTROL MILI KD CONTROL MILI KD MILI 1.51.5 ACTIN 1.5 * 1.51.5 miwi * 1.5 ** 1.01 * 1 1.01 1.01

0.50.5 0.5 0.50.5 expression (AU)

expression (AU) 0.5 ili MILI abundance MILI M piRNA expression (AU) piRNA Miwi

0.00 0.00 0 Relative miwi mRNA expression (AU) 0.00 Ctl GpM1 GpM2 Ctl GpM1 GpM2 piR-cluster 1 2 3 4 aNPCs

D DIF 0 / DIF 4 DIF 4 / DIF 7 DIF 0 / DIF 7

5

0 Logfold change -5

1 100 10000 1 100 10000 1 100 10000 Mean of normalised counts Mean of normalised counts Mean of normalised counts

E piR-cluster 1 Human Chr16 Exon1 Exon2 Mouse 1A 1B ATG Intron1 Vac14 Mouse • Chr8 Cmtr2 Exon1 Exon2 • Hdyn • CMTR2 Human Intron1 • Vac14 ATG • HDYN VAC14 • Mtss1l • Vac14 • Il34 • MTSS2 • Sf3b3 • IL34 1A 1B • Cog4 TTAAAAATTTATTAGGCAGTGTGAAATTTTCATCTCTTAGAATTAA TGCATTGGCCGGGAATCGAACCCGGGCCTCCCGCGT • SF3B3 AAGGATTGCTACTGCATTGGCCGGGAATCGAACCCGGGCCTCCCGC GGCAGGCGAGAATTCTACCACTGAACCACCAATGCT • Fuk • COG4 GTGGCAGGCGAGAATTCTACCACTGAACCACCAATGCTCCAGCTGT CCAGCTGTTTGGT • St3gal2 • FCSK GAGCAGCTGTGTGGCACCTCCCTATAACCTCATCCCTGGGCCTCCA • Ddx19a • ST3GAL2 TGTGTATATGTTGA q22.1 • Ddx19b 8qD3 • DDX19A Mouse tRNA-Gly: n-TGgcc11 (3’< 5’) Mouse tRNA-Gly: n-TGgcc10 (3’< 5’) q22.2 • Aars 8qE1 • DDX19B • Clec18a • AARS Human tRNA-Gly: TRG-GCC2-5 (3’< 5’) Human tRNA-Gly: TRG-GCC5-1 (3’<5’) • Pdpr • CLEC18C piR-cluster 1 (3’< 5’) piR-cluster 1 (3’< 5’) • PDPR Fig. S1. KD of Mili does not affect Miwi mRNA levels, and deplete piRNAs abundantly expressed in aNPCs.

Related to Figure 2.

(A) Mili mRNA (left) and protein (right) levels in aNPCs upon transfection with control GapmeR (Ctl) or two different GapmeRs (GpM1, GpM2) targeting Mili (Mili KD). (B) Expression of four of the most abundant piRNA clusters in control and Mili KD by two different GapmeRs aNPCs. (C) Relative expression of Miwi in undifferentiated aNPCs in vitro transduced with viruses transcribing a Scrambled short-hairpin (Control) and shMILI (Mili KD). (D) Pairwise comparison of 298 piRNA clusters differentially expressed in undifferentiated aNPCs (DIF0) or upon viral-induced neurogenesis (DIF4-7). (E) Chromosomal location of piR-cluster 1 in mouse and human; (Right) genomic location and sequences (underlined red text) of piR-cluster 1 corresponding to tRNA-

Gly (underlined black text). Data are expressed as mean ± SEM, n = 3 (A-C) and n = 2 (D-E) independent experiments. t-Student test as post hoc: ∗p < 0.05, ∗∗p < 0.01.

3 shMILI Nestin IF shMILI Ki67 IF Gasperini et al SupplementaryshMILI GFAP Material

shMILI WB GFAP

Scrambled shMILI Scrambled shMILI Scrambled shMILI Scrambled shMILI A Nestin Ki67 B CONTROL MILI KD 100 100 CTL KD 250250 GFAP ** ACTIN 80 80 200200 ** 2525 ** Ki67 ** 60 60 150150 2020

1515 1515 Nestin 40 40 mRNA expression (AU)

1010 1010 GFAP DAPI

20 20 expression (AU) Ki67nuclei+ (%) 55 55 Nestinnuclei+ (%) GFAP relative abundance

0 0 Relative 00 00 gfap GFAP abundance GFAP CONTROL MILI KD C 400 1 1 1 1 3 220 380 2 2 40 2 2 200 3 3 3 3 360 2.5 35 CTL CTL

CTL CTL 180 4 4 4 4 340 30 160 5 2 5 5 5 320 6 6 6 140 6 25 300

7 1.5 [TPM] 7 [TPM] 7 [TPM] 7 [TPM] 120 280 8 8 20 8 8 100 260 9 1 9 9 9 15 80 240 MILI KD MILI 10KD MILI 10 10 10 MILI KD MILI KD MILI 60 0.5 10 220 11 11 11 11 40 200 12 12 5 12 12 0 Id2 Nfix Dll1 Rb1 Gli3 App Calr Nbn Gfap Sox6 Jag1 Rhoa Pax6 Sox3 Eif4e Itgb1 Hes5 Ddx6 Trp73 S100b Meis1 Prpf19 Hmg20a Rap1gap

Fig. S2. Mili KD does not alter aNPC stemness and proliferation, and induces the expression of genes involved in astrogliogenesis. Related to Figure 3.

(A) (left) Confocal microscopy images of undifferentiated aNPCs in vitro transduced with viruses transcribing a

Scrambled (Control) and shMILI (Mili KD), immuno-stained with anti-Nestin (white), or anti-Ki67 (purple) antibodies and stained for nuclear DNA with hoechst (blue); (right) Percentage of Nestin or Ki67 positive cells over total cells. (B) Gfap mRNA (left) and protein (right) levels in control and Mili KD in differentiated cells (DIF

7). (C) RNA seq. expression data of genes involved in astrocytes development and astrogliogenesis and regulation of neuronal fate in Mili KD aNPCs DIF4 compared to Scrambled control. Data are expressed as mean ± SEM, n

= 3 independent experiments, (in C, 2 separate flow cells per sample). ∗∗p < 0.01. Scale bar 50 µm.

4 shMILI Bcl2 DIV7

Gasperini et al Supplementary Material

A DIF 4 DIF 7 CONTROL B Scrambled Caspase 3 DAPI Casp3 MILI KD shMILI 100 15 **

8080 CONTROL 10 6060

4040 expression (AU) 5

2020 Bcl2 MILI KD MILI %Caspase 3+cellsDAPI /

0 0 0 DIF4 DIF7 DIF7 A. Sh-ScrambleC Sh-MILI-KD 12 40 250 1400 10 CTL 5 20 KD 0 [TPM] 5 [TPM] 50 [TPM] 400 [TPM] Id4 Id2 Id1 Id3 Ogt Ada Atf4 Dbp Ngfr Sik1 Nfil3 Relb Arntl Sirt1 Cry1 Per3 Egr1 Per1 Per2 Rorb Ass1 Klf10 Top1 Cdk1 Ddx5 Crtc1 Ddb1 Nono Crem Nr2f6 Fbxl3 Clock Zfhx3 Prkdc Siah2 Ptgds Prok1 Arntl2 Prkg1 Trp53 Nlgn1 Ncor1 Adrb1 Creb1 Nr1d1 Adcy1 Rock2 Kcnd2 Kcna2 Spsb4 Npas2 Hdac2 Gsk3b Ep300 Prokr1 Fbxw7 Ube3a Mapk9 Kdm5c Prkaa2 Adora1 Hnrnpd Hnrnpu Gabrb3 Ppp1cc Ppp1ca Mycbp2 Fbxw11 Maged1 Kcnma1 Bhlhe41 Bhlhe40 Mybbp1a Tnfrsf11a B. Ppargc1a Sh-ScrambleD Sh-MILI-KD 2.5 16

CTL 1.5 [TPM] [TPM]

KD 0.5 2 Frk Fer Tek Mlkl Des Lpp Clk2 Pld1 Stk3 Ptk2 Alad Cinp Ism1 Lhx1 Mtif3 Lnx2 Ror1 Grk4 Asb2 Itga4 Mtap Rgs6 Sgce Rftn1 Mylip Rgs3 Nek8 Znrf3 Acsl1 Gatm Lin7a Gnb4 Ttc33 Stk10 Arntl2 Stk39 Trp73 Polg2 Snai2 Pola2 Ifnar2 Prkg1 Arfip1 Cald1 Btbd3 Ednra Lynx1 Tbx15 Msrb3 Mark1 Tbx18 Mat2b Syne2 Nxph4 Stat5a Nucb2 Apbb2 Nfatc3 Dguok Wdr53 Lgals4 Lypla1 H2-M3 Myo1b Pdlim1 Lama4 Lamb1 Zbtb7c Clec4e Ublcp1 Ptpn22 Rbms3 Rbms1 Atp1a2 Col5a2 Col5a3 Rnf167 Myom1 Tinagl1 Card10 St8sia4 Samd9l Hdac11 Capn11 C1qtnf7 Fam13c Slco1a4 Rasl11b Runx1t1 Col28a1 Atp13a4 Col27a1 Cd300lg Rasgrp2 Arhgap6 Zc3hav1 Cacna1h Cyp27a1 Fam118b Tmem173 Tmem220 Phospho2

Sh-Scramble C920021L13Rik

Sh-MILI-KD 60 15 60 400

10 40 CTL

5 20 10 [TPM] 50 [TPM] KD Irf2 Ptn F2r Sp1 Sp3 Ubb Ppic Pls3 Ets1 Abi2 Syf2 Btg1 Hsf1 Dmd Agrn Nptn Ubr2 Fosb Trib2 Ece1 Ddx5 Ptprk Aup1 Rhoa Gpc4 Nub1 Tmx3 Tcf12 Axin2 Sike1 Idh3g Ddit4l Hsdl2 Ptcd2 Pnrc1 Sept7 Sept2 Parp8 Nr1d1 Mxra8 Ephx1 Rock1 Syne1 Pofut2 Sned1 Pik3ca Amotl1 Tmod3 Dnajc4 Snapin Npepl1 Ctnna1 Pitpnc1 Mrps31 Mboat2 St6gal1 Dynlrb1 Tnfrsf21 Cyp4f13 Ppp2r5a Arhgef25 Timm17a Arhgap42 Ppp1r12a

C. 9930111J21Rik2 Sh-ScrambleE

Sh-MILI-KD 60 60 CTL [TPM] 10 [TPM] 10 KD Zfx Fry St5 Abr Inf2 Ldlr Klf4 Pltp C1rl Elk3 Itpr1 Pfkp Sirt1 Relb Ppt2 Rara Layn Tcf7 Fzd5 Ttpal Itga1 Irak2 Itga7 Dysf Shc1 Pex6 Eml2 Pex5 Kirrel Xbp1 Hps6 Nek6 Uba2 Cd44 Cnn2 Rhod Chn1 Cdr2 Myof Bicc1 Gbp2 Osmr Tbx3 Gfra2 Avpi1 Bcam Irgm2 Stk35 Nkd2 Uaca Tradd Ppard Parp3 Tcirg1 Plscr2 Adcy9 Nhsl2 Tppp3 Ndst3 Pdgfb Tgfbr2 Hdac5 Trpm4 Wnt5b Epha2 Pde4b Ggta1 Acadvl Slc2a1 Inpp4b Synpo Rassf1 Zbtb7a Tfap2a Mical1 Dnajb4 Osbpl3 B3gnt2 Col4a5 Steap4 Gpsm3 Fblim1 Pik3cd Ablim1 Lama3 Slc5a5 Ldlrap1 Unc13c Akap12 Atp2b4 Hspa1a Unc45a Hsd3b7 Hspa1b Oxnad1 Map2k3 Plekhg2 Slc10a3 Dcdc2a Sh2d3c Slc6a17 Tspan17 Nup210l Spata13 Cdk2ap2 Slc25a25 Fam118a Fam129b Serpinb6a Cdc42ep4 Sh-Scramble Adamts16

Sh-MILI-KD 20 40 60 60 60

30 CTL 10 20

10 10 [TPM] 10 [TPM] 10 [TPM] KD Id1 Bcr Jun Fos Klf9 Klf6 Eif1 Ier2 Ubc Arf6 Utrn Msn Zic2 Flna Sik1 Ing5 Ccni Dtx3 Phf1 Btg2 Rela Egr2 Per1 Egr1 Mafk Jund Junb Imp3 Pdxk Tspo Extl3 Ldha Vasp Dgkz Ptms Nck2 Pkd1 Ddit3 Ehd2 Srsf5 Lzts2 Msx1 Rhob Oaz1 Sort1 Isca1 Fosl2 Ltbp3 Tpm4 Ccnl1 Stk40 Lrp10 Siah2 Arpc5 Ndel1 Galk1 Spry4 Nat14 Hbegf Tacc1 Foxc1 Nr4a1 Socs3 Kcnk3 Adcy6 Klhl21 Anxa7 Nfkbia Anxa2 Dusp6 Nuak2 Eftud2 Abhd8 Capzb Mknk2 Dnpep Mob3a Pbxip1 Lama5 Plxnb2 Phlda1 Pttg1ip Hivep2 Dnajc3 Zfp703 Ptp4a3 Atp10a Minos1 Myl12a Csrnp1 Chmp5 Tnfaip1 Tm4sf1 Rpl22l1 Rabac1 Cdkn1a Chchd2 Prkar1a Sertad1 Plekhh3 Psmb10 Aldh4a1 Ankrd50 Arhgap1 Tsc22d3 Cyp20a1 Gadd45a Gadd45b Slc25a33 Ppp1r15a Tnks1bp1

1500009L16Rik

Fig. S3. piRNA pathway depletion does not lead to apoptosis, rather alters the expression of genes involved

in inflammatory responses. Related to Figure 4.

(A) Fluorescence microscopy images of Control or Mili KD cells 4 or 7 days after spontaneous differentiation

(DIF 4, 7), immunostained with anti-cleaved caspase-3 (green) and for nuclear DNA with Hoechst (blue). (Right)

Percentage of cleaved caspase-3+ cells over total cells. (B) Bcl2 mRNA expression level in control or Mili KD

cells 7 days after induction of spontaneous differentiation (DIF7). (C-E) Heatmap of differentially expressed

transcripts in RNA seq from Mili KD neuroblasts, involved in circadian regulation (C, upregulated)

or ROS production (D, upregulated; E, downregulated). Data are expressed as mean ± SEM, n = 3 independent (in

C-E, 2 separate flow cells per sample). t-Student test as post hoc: ∗∗p < 0.01. Scale bar, 50 µm.

5 Gasperini et al Supplementary Material

Supplementary Data Table S1.

Genomic location on mouse genome (NCBI37/mm9) and read sequence of piRNA clusters in undifferentiated aNPCs (DIF0) and upon induction of neuronal differentiation (DIF4-7).

Supplementary Data Table S2.

Genomic location on (hg38) of piRNA clusters expressed in human neural stem cells (NSC) and astrocytes.

6