Supplementary Table 3d: PCR primers for genomic sequencing-based phasereconstruction

Transcription

1 Supplementary Table 3: Detailed lists of grnas, primers, donor constructs, antibodies, Gene Expression Omnibus (GEO) identifiers for Chip-seq and DHSs datasets and CRISPR/Cas9 genome edited cell lines used in this manuscript (see Online Methods). File contains 7 Tables: Supplementary Table 3a: grnas Supplementary Table 3b: Donor constructs Supplementary Table 3c: Primers Supplementary Table 3d: PCR primers for genomic sequencing-based phasereconstruction Supplementary Table 3e: Antibodies Supplementary Table 3f: Gene Expression Omnibus (GEO) identifiers for Chip-seq and DHSs datasets Supplementary Table 3g: Summery of targeted and maintained cell lines

2 Supplementary information Table 4a: grnas Targeting approach grnavector BFP YFP EGFP EGFP BFP SNCA-Rep1- deletion SNCA-Rep1- Inron-4- Enhancer deletion Intron-4- Enhancer SeedPAM taaccaactggtactatacctatggaagac atctccattgtaaatgcttgtttcacactctg agagtgtgaaacaagcatttaagttggttaa aatggcctctgggatggtcacagtcggcaat tttcccgttacatgctctctaatttatgatg aatggattttctttcccgttaagtcggcaat EGFP Annealed Oligonucleotides 5 -caccgtccataggtatagtaccagt-3 5 -aaacactggtactatacctatggac-3 5 -caccgtgtgaaacaagcatttacaa-3 5 -aaacttgtaaatgcttgtttcacac-3 5 -caccgtgtgaaacaagcatttaagt-3 5 -aaacacttaaatgcttgtttcacac-3 5 -caccgcctctgggatggtcacagt-3 5 -aaacactgtgaccatcccagaggc-3 5 -caccgataaattagagagcatgtaa-3 5 -aaacttacatgctctctaatttatc-3 5 -caccgattttctttcccgttaagt-3 5 -aaacacttaacgggaaagaaaatc-3

3 Supplementary information Table 4b: Donor constructs Oligonucleotide to amplify targeting Targeting Donor vector Inserted sequence/snp homology TV-Rep1-257 (TC) 10TT(TC) 9(TA) 8(CA) 10 TV-REP1-fw: 5 -AAATTCCCTGCAGGCACCTGGATAA SNCA- TV-Rep1-259 (TC) 10TT(TC) 10(TA) 8(CA) 10 ATTTGGTTCCTTAATGG-3 Rep1 TV-REP1-rw: TV-Rep1-261 (TC) 10TT(TC) 10(TA) 10(CA) 9 5 -AAATTCGGCCGGCCTGCTCTTCCCT TV-Rep1-263 (TC) 11TT(TC) 9(TA) 9(CA) 11 AAAACAATTCTCTTCG-3 TV-Int4- ~gtaattagaacaatcagggcacaaactgcaag~ Enhancer-A-T TV-Int4-Enh-fw: TV-Int4-5 -AATTCTCCTGCAGGGCAGATTCCAC Intron-4- ~gtaattggaacaatcagggcacaaaccgcaag~ Enhancer-G-C ATTCACTTTTGAGC-3 enhancer TV-Int4- TV- Int4-Enh-fw: ~gtaattagaacaatcagggcacaaaccgcaag~ Enhancer-A-C 5 -AATTCTGGCCGGCCCACTGACCAAA TV-Int4- AACTCTACCTGCC-3 ~gtaattggaacaatcagggcacaaactgcaag~ Enhancer-G-T

4 Supplementary information Table 4c: Primers Aplication Primer /Probe Oligonucleotide Comments/ modification Fragment length SNCA-Rep1-fw 5 -GACTGGCCCAAGATTAACCA-3 analysis SNCA-Rep1-rev 5 -FAM-CCTGGCATATTTGATTGCAA-3 5 FAM-tagged forward pr. 5 -GTCTATTTCTCCCTTCAATCCTGTCA-3 Allele-specific reverse pr. 5 -ACAATTATAAACACATACACATCAAACAACAGTTC-3 Taqman qrtreporter seq. G- PCR 5 -CCCAAAATACGTAAAGCA-3 VIC allele SNCA-reporter SNP (rs356165) reporter seq. A- 5 -CCCAAAATACATAAAGCA-3 FAM allele N/AQuantitative Taqman qrt- Hs _g1 N/A FAM PCR GAPDH Quantitative Taqman qrt- Hs _m1 N/A FAM PCR SNCA Quantitative forward pr. 5 -GGTGAGAATTAAAATAAAACGTTATCTCATTGCA-3 Taqman qrt- reverse pr. 5 -CTTTGTGTCAGTTCTTAATTCATGTTGCT-3 FAM PCR reporter seq. 5 -ATCCCATCTCACTTTAATAAT-3 SNCA-3 -UTR Intron-4-enhancer targeting genotyping SNCA-Rep1 targeting genotyping ChiP-qRT-PCR Quantitative SYBR green qrt-pcr 5 -AGCTACATGACTGGCCCA-3 5 -CTGAAGCCCACAGAGTGTGA-3 5 -AGTCTGACCACCATGGAAGC-3 5 -GGTTTCGTTGGAATCTGGAA-3 5 -GCAATCCTAGCAAACTACTACATCAGG-3 5 -CAGCTTAGAATGAAAGTCCTACAGGG-3 5 -CGCGCTAATGCACGTAAATA-3 Intron-4- Enhancer-SeqtwA Intntron-4- Enhancer-SeqrwA Intron-4- Enhancer-SeqtwB Intntron-4- Enhancer-SeqrwB SNCA-Rep1-SeqfwA SNCA-Rep1-SeqfwA SNCA-Rep1-SeqfwB SNCA-Rep1-SeqrwB 5 -CTGCTTGGTTTTCCCTTTTG-3 ChIP-SNCA-int4-5 -CTGCTTGGTTTTCCCTTTTG-3 Enh-fw ChIP-SNCA-int4- Enh-rw 5 -CTGCAAACGCTTCTGTTTTG-3 ChIP-SNCA-negfw 5 -AAACGTTGGCATGGAAGTGG-3 ChIP-SNCA-negrw 5 -ATACCTAGGAGTGGGATCGC-3 ChIp-SNCA- 5 - CTGGCCCAGAATCTAACAGC -3 3 UTR-Enh-fw ChIp-SNCA- 3 UTR-Enh-rw 5 - GGGCTCAAGCAAGAACAGAG -3 ChIP-Chr8- negcontrol-fw 5 - TCCTGGGTAGGAACCAGTTG -3 ChIP-Chr8- negcontrol-rw 5 - ACTCACCAAGAGCTCCTCCA -3 RPLP0-fw 5 - GCTTCCTGGAGGGTGTCC -3 RPLP0-rw 5 - GGACTCGTTTGTACCCGTTG -3 MAP2-fw 5 -TTCGTTGTGTCGTGTTCTCA-3 MAP2-rw 5 -AACCGAGGAAGCATTGATTG-3 SNCA-fw 5 - TATGCCTGTGGATCCTGACA -3 SNCA-rw 5 - TCAAGAAACTGGGAGCAAAGA-3 GFAP-fw 5 - CACCACGATGTTCCTCTTGA -3 GFAP-rw 5 - GTGCAGACCTTCTCCAACCT -3 Deletion screening Outside targeting homology Deletion screening Outside targeting homology

5 Supplementary information Table 4d: PCR primers for genomic sequencing based phase-reconstruction Primer SNCA-ph-A-fw1 SNCA-ph-A-fw2 SNCA-ph-A-rw SNCA-ph-B-fw SNCA-ph-B-rw SNCA-ph-B-fw SNCA-ph-B-rw Oligonucleotide 5 -GGTTTCGTTGGAATCTGGAA-3 5 -TTGGCATCTCCAAGGGTATC-3 5 -AGGCCCAGAGACTGATCTGA-3 5 -GGGAAGGCACCCTCTAGGTA-3 5 -ACTAGGCCCTATCCCTGGAA-3 5 -ACTGAGGGAGAGCAAGTCCA-3 5 -GCACCCATGGGGAGTTAATA-3 Fragment length A1: ~ 8.2 kb A2: ~ 7 kb ~ 13.8 kb ~12 kb Included heterozygous SNPs rs (A/G) rs (C/T) rs (A/G) rs (A/G) rs (C/T) rs (A/G) rs (C/G) rs (C/G) rs (A/G) Comment SNCA-ph-A-fw2 is in deletion of Int-4-Enhancer rs is reporter SNP

6 Supplementary information Table 4e: Antibodies antigen provider catalogue number use LMX1B E(N-14) Santa-Cruz sc-21231x ChIP-qRT-PCR LHX4(K-16) Santa-Cruz sc-22136x ChIP-qRT-PCR LHX2 (C-20) Santa-Cruz sc-19334x ChIP-qRT-PCR normal mouse IgG Santa-Cruz sc-2025 ChIP-qRT-PCR normal goat IgG Santa-Cruz sc-2028 ChIP-qRT-PCR normal rabbit IgG Santa-Cruz sc-2027 ChIP-qRT-PCR normal rabbit IgG R&D AB-105-C ChIP-qRT-PCR Engrailed-1 Developmental Studies Hybridoma Bank 4G11-C ChIP-qRT-PCR OTP Novus NBP ChIP-qRT-PCR LMX1 (Lmx1a) Millipore AB10533 ChIP-qRT-PCR NKX6-1 Sigma HPA A71678 ChIP-qRT-PCR/ Western blot Emx1 LSBio LS-C16775 ChIP-qRT-PCR Emx2 LSBio LS-B3481 ChIP-qRT-PCR/ Western blot Engrailed-2 R&D MAB2600 Clone # ChIP-qRT-PCR H3K4me1 abcam ab88895 ChIP-qRT-PCR H3K27ac abcam ab4729 ChIP-qRT-PCR/ChIP-seq MAP2 Encor biotechnology, Inc CPCA-MAP2 Immunostaining NeuN Millipore MAB377 Immunostaining TUJ1 Convance MMS-435P Immunostaining GFAP DAKO z0334 Immunostaining vglut1 Synaptic Systems Immunostaining NKX6-1 Developmental Studies Hybridoma Bank F55A10 Immunostaining LAMIN A/C abcam ab Western blot EMX2 abcam Ab Immunostaining DDK (FLAG) Origene Clone OTI4C5, Western blot TH Pel-Freeze P Immunostaining

7 Supplementary information Table 4f: Gene Expression Omnibus (GEO) identifiers for Chip-seq and DHSs datasets Sample H3K27ac ChIP- H3K4me1 ChIP- H3K4me3 ChIP- Seq Seq Seq DHSs-Seq BI_Brain_Angular_Gyrus GSM GSM GSM GSM GSM N/A BI_Brain_Anterior_Caudate GSM GSM GSM GSM GSM N/A BI_Brain_Cingulate_Gyrus GSM GSM GSM GSM GSM N/A BI_Brain_Hippocampus_Middle GSM GSM GSM GSM GSM GSM N/A BI_Brain_Hippocampus_Middle_150 N/A GSM GSM N/A BI_Brain_Inferior_Temporal_Lobe GSM GSM GSM GSM GSM N/A BI_Brain_Mid_Frontal_Lobe GSM GSM GSM GSM GSM GSM N/A Bi_Brain_Substantia_Nigra GSM GSM GSM GSM GSM GSM N/A Fetal Brain N/A GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM GSM

8 Supplementary information Table 4g: Summery of targeted and maintained cell lines Cell line Genetic modification/ Genotype Number of maintained clones WIBR3 hesc Wild-type NA BGO1 hesc Wild-type NA Experiment displayed in manuscript Comments Parental hesc lines WIBR3-NURR1-EGFP 1 All subsequent cell lines In-frame of EGFP are derived from these BGO1-NURR1-EGFP into NURR1 locus 1 sub-clones WIBR3-SNCA-Rep1 deletion BGO1-SNCA-Rep1 deletion ΔRep1/Rep1-261 ΔRep1/Rep WIBR3-SNCA-Rep1 BGO1-SNCA-Rep1 WIBR3 homozygous enhancer deletion WIBR3 Homozygous deleted subclones WIBR enhancer In Phase with A-(FAM)- reporter SNP Rep1-257/261 2 Rep1-259/261 3 Rep1-261/261 2 Rep1-263/261 2 Rep1-257/261 2 Rep1-259/261 2 Rep1-261/261 2 Rep1-263/261 2 ΔE4/ΔE4 4 ΔE4/ΔE4 3 A-T/ΔE4 4 G-C/ΔE4 3 A-T/ΔE4 3 A-T/ΔE4 2 Extended data Fig. 10 Fig 2c,d (1 clone) Extended data Fig. 4, Fig 5h-j Fig. 2 c,d Extended data Fig 4, Fig 5a-g Fig. 2 c,d Extended data Fig 4, Fig. 5a-g, Fig 7 Non-targeted subclones from enhancer- experiment as control