Figure S1

Fig S1. Design of the photolithography masks. A. Layout is based on (Taylor 2010). Changes have been introduced in many dimensions to increase the overall yield when cutting the synaptic compartment. The image shows the Mask containing the reservoirs as well as the perfusion channel, for second exposure. Height of the photoresist (SU-8-2050) for this step is 160µm. B. Mask containing the microgrooves, used for first exposure. Height of the photoresist (SU-8-2025) for this step is 20µm.

Figure S2

YPEL5

CYP46A1 CNIH2

DDX42 B PBX1 CNR1 CSMD1 CHST2

MBNL2 CSPG5 CFAP54 ENSA A CPLX2 NTRK3 HDGF RAP1GAP2 CEP170B COL27A1 BOD1L PHIP FAT3 ARID4B D5ERTD579E RASGRF1 LARS2 CAMKV ABHD4

PNMAL2 SLC2A3 TAOK2

BRIP1OS SLC4A3 AAMP RAB10OS ATP6V0E2 RALGAPA1 BEX3 SCAMP5 RAPGEF1 TMEM30A ACSL6 UHMK1 SAT1 RBFOX3 SNHG20 TMEM132B SPOCK2

SLC12A5

NRXN2 YPELRGS7B3 P COPA

RSF1 DCAF7 FABP7 RAB3GAP1 CFAP36 LSAMP PLATR28 PALM ZBTB38 MT-TQ WIPI2 MPDZ CELSR2 TENM3 FEZ1 PRPF4B UBAP2L mt-Rnr2 DCDC5 CDH23 LRP1 TSC22D4 STOX2 HECW1 ARFGEF3 WDR1 T2 CHST1 ATP6V1G2 TLN2 IMMT WDR26 ABLIM2 WBP2

ATP6V0B TNKS2 CACNA1C RNF10 ATP6V1E1 ARHGEF2 MEIS2 RAB15 HECTD1 TSPAN7 ARPP21 BIRC6 CWC22 MT-RNRSY1T11 RAB1B PPIG MAGI1 KAT6B NAP1L5 GABARAPL1 SF3B1 PAFAH1B2 EML5 TACC1 MTUS2 HMGCS1 CDC42BPA MTPN CACNA1BSLTM NDRG4 RAB10 FAM234B CCDC93 ATP6V1C1 CDK5R2 TENM4 PRPF8 BCAT1 mt-Rnr1 CDK16 GRIA2 RGS7 APC CLSTN2 GNB1 IQSEC1 DCLK1 FIP1L1 NEDD4 USP19 DMXL1 USP22 ATP2B1 ATP6V0A1 SYNE2 AKT3 ATP6AP2 ATP1A1 GLS TTN PSAP ANAPC5 TLE3 RNY3 PITPNM1 NKTR ADD3 CYP51 ATXN7L1OS2 RBBP6 MIAT DNAJC11 SFPQ RABL6 DDX17 NAV2 OXCT1 SRCIN1TAGLN3 GDI2 SNRPN RN7S2 ADD2 TJP1 MATR3 PTPRD PRICKLE2 MCF2L PRDX6 CHCHD2 ALS2 PLCB1 CAP1 TET3 GDI1 DNAJA2 CFL1 SNHG1 TAF1 RYR1 CBX6 ADGRV1 NSD1 GLYR1 NDFIP1 FBXO44 CLIP3 PSD3 ANK HSPA9 RAB6A CAPZB AUTS2 CDC42SE2 MT-RNR2 ATP1A2 SQLE DMXL2 CDS2 MAPK6 KCNQ2 WNK1 D10WSU102E ATP1B1 TNIK TAOKCTS1 D SDHA CT010467.1 MAP1LC3B UBR3 BCLAF1 MT-ND2 HSBP1 PFKM SPTBN1 ARHGEF9 KIF21B GSK3B EXOC4 SULT4A1 HSP90AA1 ATP1A3 DOCK9CDK17 FTO ATP6V1A FDFT1 TPM1 SLC5A3 PPP1R9A PPP2R2C SMC1A ZHX3 HUWE1 KCNH7 GRIA1 ABR CAPZA2 CLCN3 RAB7 ATP2C1 PPT1 WDFY3 GRIA3 TRPC4AP HNRNPM EWSR1 ATRX CDK14 TRRAP MT-COAD1 ORA1 SLC7A14 MICAL3 LAS1L WT1OS BSG CLCA4C-PS CSPP1 ATP9A HSPA4 PLK2 PLS3 NDUFS1 CDIP1 DYNC2H1 TSC22D1 DTNA COPG1 MAST2MT-ND4 PPP1R9ZMYBND11 CHD9 PPP1R12B FKBP8 OPA1 MT-ND6 6 TMEM178B ARID1A TRIO UBE2D3 SRGAP3 LNCENC1 ARF3 ATP1B2 LUZP1 YLPM1 CLSTN3 GLG1 NAV1 DGKZ SNHG11 EIF4A2 RELN SQSTM1 NFIX ZFR 193 ATP6V1D ZRANB2 BRD2 TAX1BP1 FRY PTPRK SKP1A SHANK2 CLSTN1 SLC38A2 TBC1D14 ADIPOR1 ADGRL3 PAFAH1B1 AFF4 CTNND2 IPO9 Rny1 GPI1 PCDH15 PTMS TRAK1 AARS SYT4 RRP1 ITPR1 H3F3B TUG1 VPS35MEG3 CADPS NUDC GOLGB1 CNBP ANKRD11 MKRN1 SORL1 ASNS NDUFS2 SUB1 HOMER1 ATP6V1B2 ABAT NIPBL SEPT7 ZWINT SH3GLB2 CDC42 ARHGEF4 DST GRIN2A MSMO1 CEP83OS CTTN PKM HSPH1 DLAT HP1BP3 VLDLR MAP4K4 MGRN1 ATP2B2 RABGAP1L RBFOX2 SEPT11 AKAP13 GAS7 ABCA2 SARS MYO18A TTLL5 CUX2 ATRN LRRC58 SIPA1L1 PDLIM7 SEPT5 PUM1 PSAT1 MRFAP1 STRAP UBA1 PDE4D USP24PSIP1 SCN8A SNRNP200 ANK3 CBX5 ANKRD13C FIRRE TCF4 ENO2 HDLBP ACLY ZMYND8 ARHGEFFTH7 1 CALM1 SUPT5 AGAP2 TMOD2 SEPT8 SDC3 EXOC6B ELMORANBP2 2 LRP2 HYDIN PAK1 ATP6AP1 ZMAT2 ENO1 ATXN2 LPGAT1 PCNX DMTN SRGAP2 PPP1CB BAP1 DDX3X RAB3GAP2 MORF4L1 SSH2 GRM5 CEP350 ZC3H13 AMPH PURB MT-ND1 NEDD4L MED13L SLC6A17 FKBP1A FTL1 LSS TRAK2 LAMP1 OGT CPLX1DIP2B ERC1 PVT1 AGPAT3 FRYL CCAR1 CKB LMTK2 Rny3 GRIN1 DNAJB6 BAZ2A NCOR2 TOM1LNE2FM PIN1 BRWD1 ARPC2 EP400 MT-ND5 MT3 EEF2 SERINC3 NCL CAMK2N1 TNR SMC3 GBP11 PFKP CLPTM1 ARHGDIHAIVEP2 MAGIYBX2 1 SEL1L MAT2A COX5B IDH3B DCTN1 UBE3A ATP2A2 UQCR10 CAMTA1 PHLDB1 SF3B3 DDX46 LRRC7 RAB11FIP4 PEAK1 NHSLGRAMD12 B MT-CYTB SV2B TRP53COR1 SAFB DBN1BASP1 DROSHA ARHGAP32 MPRIP ANK2 ASTN1 ADGRB2 RBM46OS KMT2E GAPVD1 CLU HSP90AB1 KPNA1ADD1 NARS TRIM2 UBR5 JMJD1C PTK2B RNF157 GOLGA4 DCTN2 ABI2 CHD8 SYT1 PRDX2 UQCRH PHF3 SCD1 ACTB PPP1CA ABCF2MYCBP2 SUCLA2 SOX2OT EIF3C FLNA CLASP1 D6ERTD474E EIF5 PPM1E NBR1 UHRF1BP1L PCMT1 CCNI NME9 CAMK2A CCDC47 PRDX5 CSMD2 NUMA1 OGDH MRPS6 ARRB1 SUMO3 CAMK2B ADGRL1 SRRM2 GDE1 DNAH9 NF2 PGRMC1 NRXN3 PSMD1 AKAP6 MAPRE2 DDX24 SLC25A5 RAB6B OSBPL1A KIFC2 SODIST2 1 ANKS1B CHD2 SHANK1 DLGAP1 PDAP1 GRIN2B ANKRD46 GARS NDUFA10 Rpph1 AU022754 DNAJC5 CYC1 PDS5B SYNE1 PLATR21 CKAP5 SPTB LRPAP1 PPP2CA MYL12DYNC1LIB 2 MYO6 PIK3R2 RNY1 CYFIP2 UGGT1 PHC3 RYR2 GSTM1 ERC2 KCNB1 SORBS2 GNG3 GSN WASHC2 PRKCB ZEB2GOT1 PPIP5K1 GPRASP1 FYN MALAT1 OPCML RAB14 IGFBP2 STMN1 CHKA NDUFV2 ANKRD12 INSR CTNNB1 UBE2O PCDH10 DIRAS2 NEB CCTA5RHGEF17 SPTAN1 TCERG1 RNF20 DNAH1 TECR CLIP1 HSPA8 CASC4 APBA1 CS EIF4G3 PRKAR2A SUPT16 GNAI2 NDUFV1 ARF1 FBXW7 GABBR1 SF3B2 MIB1 PTPRT HABP4DDX5 TANC2 CHGA LRP8 TOP1 KCNJ10 HNRNPR ATF7IP ADAM22 DPYSL2 SEL1L3 SREK1 PRKCE ACTG1 PSMD4 MAPRE1 SLC4A4 NDUFB8 ZFP451 TRERF1 BPTF PTEN MADD NTM DLG4 ADGRB1 SLC25A4 PIP5K1C CACNA1A RN7S1 FBXO2 FBXL16 SLC6A6 IQSEC3 COX7B UQCRQ NPTN IPO7 SBNO1 EIF5A SON DENND4A FNBP1L MORF4L2 NEFL PCYT2 AHCYL2 PHF20 SRPK2 EIF5B GIGYF2 CTSL HSPA5 ACACA EPRS CACNA2D1 KMT2D DDN ACTR1B KIF5B SYNGAP1 FRMD4ANCKAP1 INO80DOS USP9X MDGA1 NDUFA13 EPHA4 RAPGEF4 CIT SETD5 NACA SLC17A7 GFAP GPATCH8 GNL3L GTF3C1 SEPT3 TTLL7 CELF1 CREBBP KMT2C SNHG17 COX6A1 PDHA1 AIRN KCTD17 CALM2 NCOR1 SLC3A2 ZFP106 BAZ2B DDB1 IKBKB PPFIA3 MALAT1 ANKRD17 RNF14 TSPOAP1 PPM1L SRRT RSRP1 XPR1 EPB41L1 DLG3 HNRNPC DLG2 UQCR11 SPTBN4 NDUFBTMCC9 1 NSF CHGB EIF4H KCNQ3 DLGAP4 ZFP148YWHAB SF1 MTOR FAM171B SNHG12 RIMS1 FTX AAK1 HSPD1 PACSIN1 GAS5 TIAM1 CCND2 ABCF1 PSMC1 ATP5A1 LRBA ACTN4 PABPC1 SGTA UQCRC1 MBP EEF1A1MINK1 NRXN1FAM81A FASN COX4I1 AHCYL1 AP2B1 TANC1 ATP5C1 CLTC KMT2A PHACTR1 AKAP9CDKL5 MECP2 TRIM44 ITPR2 PEBP1 PNISR DNAH10 KCNMA1 KIF5A PTPRA TRP53BP1 CALM3 TUBB4B MAPK10 THRA GPX4 MIR99AHG ATP5J RTN3 THOC2 KAT6A SCD2 PLB1 ABHD2 HCN1STMN4 ABCC5 ASPH RAB3A PTPN11 UBB GNAO1 AKAP8LEIF3B HERC2 DNMT3A PLCG1 CDC42SE1 PRDX1 PTK2 TRIM9 CNOT1 ITSN1 SMG1 FBXW11 LUC7L3 PSMC5 EIF3A CAMTA2 SLC4A10 ATP8A1 CSNK1A1 DPP6 MYO10 MDN1 RMRP_2 SOX1OT YARS SLC6A1 OXR1 IDH1 AGAP1 VGF ACOX1 APOE DLG1 RNF220 RTN1 BRINP1 TUBB2A ATPHOOKIF1 3 PTPRS SPEN YWHAE SNX3 TMEM59 ARNT2 TTC3 SRSF11 OAZ1-PARHGAP2S 6 ADCY1 IDH3A SLK AGO2 GOLGA2 SCG2 ACAP3 PPP1R12A CELF2 UTRN ATP5O STMN2 STAT3 DMD CCT7 TRIM35 TPR EPB41L3 PSMC3 KDM5B SPAG9 ARHGAP20OS DYNC1H1 USFCSDE3 1 HIP1 SDCBP MT-TM TUBB5 DAB1 ZYG11B CACNB4 PTPRN CALR RABGAP1 NDUFA4 SERINC1 PREX1 GABBR2 HECTD2OS FAIM2 STX1B KIFAP3 ACIN1 GPM6B SRSF5 TRO RANGAP1 PDE4DIP COX6B1 HELZ TOP2B ROCK2 FRAS1DDX1 PLD3 CHD6 ST13 SYNJ1 PCSK1NDOCK3 EIF4G2 ATP5B CAMK2D CACNA2D3 YWHAZ FUBP1 SSPO SPHKAP OBSCNTMSB4X MAP1B ALDOA SLC38A1 CTTNBP2 JAKMIP1 VCP AFDN KIF3A RUNDC3A PSMD2 MAP1A MACF1 SLC25A2ILF3 2 USP5 MAPK9 SMARCC2 MGLL CLASP2 NELL2 NBEA CADM1 ARHGAP21 ICE1 NISCH RAB2A VSNL1 MUC16 KIDINS220 SUPT6 SNAP91 PRRC2C SPTBN2 RPPH1 LUC7LTNS2 1 CELF4 RABEP1 SNAP25 SCN2A RIAN RBM5 R3HDM2 RPL4 FAT1 ALDOUNC13C B MEGF8 PHF24 PREPL PUF60 DISP2 STRN4 HMCN1PPP2R1A APBB2 KIF21A GPD2 BECN1 MYO5A GHITM MKLN1 EIF1 IWS1 MAP2K4 VPS41 NRD1 RBM6 CLTA MYH10 ATXN1 DAG1 PIKFYVE ARL6IP1 PDIA3 MAPT MYL6 PPFIA2 PLEC ATXN7L3B NRCAM HERC1 PRNP HNRNPULGRM2 7 PSMC4 ATP13A2 STXBP1 RTN4 YWHAG PIEZO2 TSPAN3 SORCS2 ZBTB20 TCF25 HTT SGIP1NMT1 GAD1 ASH1L MCC DGKD KALRN PPP3CA SDK1 PRRC2B CCT6A RAB31 EPN1 PSMB6 DGKG STRBP NAP1L1 RIMS2 VAMP2 NAPA SLC4A8 MEF2C AP1B1 HNRNPU TMBIM6 DNM1 TSPAN5 ATF4 SETD2 CHD4 PTPRN2 CADM2 MTCL1 TACC2 DCAF8 PTPRG OS9 LRP1B SNRNP70 SERBP1 ATP5G1 HDAC4 CRMP1 ACO2 SCN1A SNAP47 TRANK1 RASAL2 ODF2 NCOA2 RBBP4 RBFOX1 ABLIM1 LDHA GPX1 KIF3C JPH3 JAK1 NF1 ATP5J2 LDHB USH2A RBM39 KIF1B SPOP CPSF6 MRAS MAPK8IP2 GABRB1 RPSA MAST4 PPP3R1 AKTIP MROH2A CLTB EPB41LC2OX6C PCDH9 ARHGAP35 CNTN1 DNAJA1 ENAH PCLO CDC42BPB USP34 TPT1 CACNA1E HDAC9 PPIA KLC1 HNRNPA3 SRRM1 SYNGR1 RPH3A RYR3 TRIP12 LASP1 HNRNPA2B1 HIVEP3 ETNK1 MYO9A ATP5H DNAJC13 RPN1 UNC80 SNHG15 ARL8B QK MLEC CHN1 TNRC6B TMEM250-PS NSMF OAZ1 MYT1L SPIRE1 BTBD3 CTSB CAPRIN1 RPTOR MAP4 HPCA SMARCA4 HK1 MAPK1 NEAT1 GRIPAP1 FAM49A TUBB4ACRYAB CAMSAP2 GLUL MDH2 TM9SF3 DNAJC6 KSR2 TUBA1A CALN1 UNC13A PRRC2A COX7A2 DNAH2 TEF CANX PLP1 NAPB FGFR1OP2 DNAH8 FRRS1L CADPS2 PCM1 XPO6 EEF1A2 SCAMP1 TECPR1 GATAD2B EIF4G1 SPECC1 NFASCSMARCA2 D1ERTD622E CRY2 SYP MAPK8IP3 PI4KA ROBO2 AP2M1 TULP4 INPP4A RHEB KIF5C TUBA4A UCHL1 CHD5 ACSBG1 PIK3R1 CHL1 TPI1 HSP90B1 ATXN10 ZAN EFR3B AP3B2 UBR4 FLOT2 POU3F3 MARCH6 ATP10D NTRK2 PSMD7 MAP2 MYH9 NFE2L1 DYNC1I2 CDH13 BAZ1B NSD2 SUDS3 TPPP HNRNPK NAV3 NCAM1 EPS15 CHD3 HPCAL4 CARS EP300 DYNLL1 CACNA1I LIMCH1 PRKACB JARID2 NKX2-2OS_2 BC002059 APBB1 DNM3 APLP2 ADAM23 VEZT DYNLL2 ZFP638 SPON1 YWHAH CABIN1 APPL1 GABRA1 APLP1 OLIG1 IGSF8 BRAF RBM25 CUX1 SNCA DPP8 DIP2C PFN2 SYN1 GAA MLF2 PARP6 ADARB1 OSBPL6 HSPA4L SLC23A2 NRSN1 DHX30 AKAP11 UTP20 PJA1 AP3D1 BSN GAPDH NUDT3 VPS13D EPHA5 PTPRZ1 CCDC88A CPNE6 APBA2 PRKAR1A HECTD4 PCYOX1 PJA2 APP SFI1 SERPINE2 SACS NUDCD3 ARAF TOLLIP

KIF1A FAM168A PCMTD1 STMN3 FAM126B ABCA4 PLATR20 PRKAR1B SYN2NCAN

CDK5R1 PPP4R1L-PS SYNGR3 OLFM1 CD81 RC3H2 SLC22A17 GTF2I PEA15A

SLC24A2

USP47 LNCPINT MAGED1 MROH1

GABRB3

ITM2B C NDRG3 1423 mRNA 116 lncRNA Oxidative phosphorylation

Synaptic vesicle cycle

Long-term potentiation

0 5 10 15 20 25 -log10 (p-value)

HECTD2OS

D TRERF1 TMEM250-PS

MEG3 NEAT1 MMU-LET-7G-5P

RN7S1 RIAN TUG1 OAZ1-PS MALAT1 MT-RNR2 MT-RNR1 MT-TQ MMU-MIR-6240

DLEU2 FTX MMU-MIR-2137 RPPH1 SNHG12 PPP4R1L-PS VMN2R-PS69 RMRP_2 GAS5

SOX1OT

Fig. S2. Network analysis of synaptic lncRNAs. A. Venn diagram showing that the 6 annotated lncRNA detected in synaptosomes (red circle) are part of the 199 lncRNA detected in the microfluidic chambers. The 6 common lncRNAs are listed in the left panel. B. Out of 1460 mRNA detected in microfluidic chambers, 1423 could be fit into a network that also contains 116 of the

199 annotated lncRNA. Specifically Malat 1 appears as a hub lncRNA. C. Three main pathways represented by the synaptic mRNAs potentially controlled by the 116 lncRNAs. D. Network showing the interactions of synaptic lncRNA (green) and microRNA (blue) detected in microfluidic chambers. Note that the two well-studied lncRNAs Malat1 and Neat 1 are part of a network that controls microRNA-6240, let-7G-5p and microRNA-2137. Please note that panels B and D are available as searchable high resolution PDF files as additional supplemental data.