title: Pool_520_AD010_indexed


title: Pool_521_AD008_indexed

status: Public on Aug 10 2017

type: SRA

city: La Jolla

first: Joseph

middle: R

last: Ecker

state: CA

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

country: USA

institute: HHMI-Salk-Institute

library_source: genomic

data_processing:
genome build: mm10
For multiplexed single nucleus methylome samples, reads were first trimmed to remove sequencing adaptors using Cutadapt 1.11 with the following parameters in paired ends mode: -f fastq -q 20 -m 62 -a AGATCGGAAGAGCACACGTCTGAAC -A AGATCGGAAGAGCGTCGTGTAGGGA. For singleplex samples, -m parameter was set to 40. For multiplexed samples, 16bp was further trimmed from both 5’- and 3’- ends of R1 and R2 reads to remove random primer index sequence and C/T tail introduced by Adaptase, with the following parameters: -f fastq -u 16 -u -16 -m 30. For singleplex samples, 10bp was trimmed from both 5’- and 3’- ends of R1 and R2 reads with the following parameters: -f fastq -u 10 -u -10 -m 30. Trimmed reads for mouse and human single nuclei were mapped to mm10 and hg19 reference genome, respectively. R1 and R2 reads were mapped separately as single-ended reads using Bismark v0.15.0 with parameter --bowtie2. --pbat option was activated for mapping R1 reads. Resulted bam files were sorted using samtools 1.3 sort, followed by removal of duplicate reads using Picard 1.141 MarkDuplicates with the option REMOVE_DUPLICATES=true. Non-clonal reads were further filtered for minimal MAPQ = 30 using samtools view with option -q30. To prevent regional mCH level estimation from being skewed by rare reads failed to be bisulfite converted, reads with read-level mCH level greater than 0.7 were excluded. The calling of unmethylated and methylated base calls were performed by call_methylated_sites of Methylpy (https://github.com/yupenghe/methylpy/) (1–3).
Supplementary_files_format_and_content: tab delimited text files of methylcytosine calls; columns in allc files are: column 1 - chromosome; column 2 - position; column 3 - strand; column 4 - class; column 5 - mC reads; column 6 - total reads; column 7 - methylated (Boolean value indicating the result of statistical test for methylated cytosines)

molecule: genomic DNA

organism: Mus musculus

source_name: Mouse frontal cortex

growth_protocol: For the production of single neuron methylomes from layer dissected mouse frontal cortex tissue, eight week old C57BL/6J male mice were purchased from Jackson Laboratories, Bar Harbor ME, and allowed a week of acclimation in our animal facility with 12 h light/dark cycles and food ad libitum, before dissections. The human brain specimen was obtained from the NICHD Brain and Tissue Bank for Developmental disorders at the University of Maryland, Baltimore, MD. The frozen middle frontal gyrus tissue belonged to a 25-year-old Caucasian male (UMB#4540) with a PMI of 23 h.

extract_protocol:
A detailed protocol for MethylC-seq libraries preparation can be found in Urich et al., (2015). MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nat Protoc 10, 475-483.
Nuclei isolation from mouse and human cortical tissues was performed as described in (Lister et al., 2013) with the following modifications: Proteinase inhibitor (11836153001, Roche) and RNAse Inhibitor (30 U/ml, PRN2611 from Promega) was added to the lysis buffer and sucrose gradients. After centrifugation, nuclei were resupended in 0.5% BSA (AM2616, Ambion) PBS (Ca2+ and Mg2+ free, 14190-144 from Life Technologies,) with protein and RNAse inhibitors. Isolated nuclei from mouse and human tissues were labeled by incubation with 1:1000 dilution of AlexaFluor488 conjugated anti-NeuN antibody (MAB377X, Millipore) at 4°C for 1 hour. Nuclei isolated from CLSun1-G35-Cre line were incubated with AlexaFluor647 conjugated anti-NeuN antibody (anti-NeuN antibody MAB377 labeled using Apex Alexa Fluor 647. A10475, Life Technologies) and AlexaFluor488 conjugated anti-GFP antibody (A21311, Life Technologies). Fluorescence activated sorting of single nuclei was performed using a BD Influx sorter with an 85 µm nozzle at 22.5 PSI sheath pressure. Single nuclei were sorted into each well of a 384-well plate preloaded with 2 µl of Proteinase K digestion buffer (1 µl M-Digestion Buffer, 0.1 µl 20 µg/µl Proteinase K and 0.9 µl H2O). The alignment of receiving 384-well plate was performed by sorting sheath flow into wells of an empty plate and making adjustment based on the liquid drop position. Single cell (1 drop single) mode was selected to ensure the stringency of sorting.
Steps of library preparation prior to SPRI purification were performed in a horizontal laminar flow hood to minimize environmental DNA contamination. Bisulfite conversion of single nuclei was carried out using Zymo EZ-96 DNA Methylation-DirectTM Kit (Deep Well Format, cat. #D5023) following the product’s manual with reduced reaction volume. 384-well plate (ThermoFisher Armadillo PCR Plate cat. # AB2384) containing FACS isolated single nuclei were heated at 50°C for 20 min. 25 µl CT Conversion Reagent was added to each well followed by pipetting up-and-down to mix. Plates were treated with a following program using a thermocycler: 98°C for 8 min, 64°C for 3.5 hours and 4°C. Each well of Zymo-Spin™ I-96 Binding Plates was preloaded with 150 µl M-binding buffer. Bisulfite conversion reactions were transferred from 384-well plates to I-96 Binding Plates followed by pipetting up-and-down to mix. I-96 Binding Plates were centrifuged at 5,000g for 5 min. Wells were washed with 400 µl of M-Wash Buffer followed by centrifugation at 5,000g for 5 min. 200 µl of M-Desulphonation Buffer was added to each well and was incubated for 15 min at room temperature before removed by centrifugation at 5,000g for 5 min. Each well was then washed with 400 µl of M-Wash Buffer for two rounds. 12 µl of M-Elution Buffer was added to each well and was incubated for 5 min at room temperature. I-96 Binding Plate was placed above a 96-well PCR plate (Applied Biosystems MicroAmp® EnduraPlateTM cat. # 4483348) and was centrifuged at 5,000g for 3min. 9 µl of eluted DNA was commonly collected in each well of the PCR plate. 1 µl of 5 µM P5L-AD002-N9, P5L-AD006-N9, P5L-AD008-N9 or P5L-AD010-N9 indexed random primer was added to each well of 96-well plate followed by mixing with vortexing. All DNA oligos were purchased from Integrated DNA Technologies (IDT). Plate was heated at 95°C for 3 min to denature the sample and was immediately chilled on ice for more than 2 min. 10 µl enzyme mix containing 2 µl of Blue Buffer (Enzymatics cat. # B0110), 1 µl of  10mM dNTP (NEB cat. # N0447L), 1 µl of Klenow exo- (50U/µl, Enzymatics cat. # P7010-HC-L) and 6 µl H2O. After mixing with vortexing, plate was treated with a following program: 4°C for 5 min, ramp up to 25°C at 0.1°C/sec, 25°C for 5 min, ramp up to 37°C at 0.1°C/sec, 37°C for 60 min, 4°C. 2 µl of Exonuclease 1 (20U/µl, Enzymatics cat. # X8010L) was added to each well followed by mixing with vortexing. Plate was incubated at 37°C for 30 min and then 4°C. 17.6 µl of home-made SPRI beads was added to each well. Sample/bead mixture from four plates indexed using distinct indexed random primers were combined followed by pipetting up-and-down to mix. Sample/bead mixture was incubated at room temperature for 5 min before placed on a 96-well magnetic separator. Supernatant was removed from each well followed by three rounds of washing with 180 µl of 80% ethanol. After air drying beads at room temperature, 10 µl M-Elution buffer was added to each well to fully resuspend the beads. Eluted sample was transferred to a new 96-well PCR plate. PCR plate was heated at 95°C for 3 min to denature the sample and was immediately chilled on ice for more than 2 min. 10.5 µl Adaptase (Swift Biosciences) mix (2 µl Buffer G1, 2 µl Reagent G2, 1.25 µl Reagent G3, 0.5 µl Enzyme G5, 0.5 µl Enzyme G6 and 4.25 µl M-Elution buffer) was added into each well followed by mixing with vortexing. Plates was incubated at 37°C at 30 min and then 4°C. 30 µl PCR mix (25 µl KAPA HiFi HotStart ReadyMix, KAPA BIOSYSTEMS, cat. # KK2602, 1 µl 30 µM P5 indexing primer and 5 µl 10 µM P7 indexing primer) was added into each well followed by mixing with vortexing. PCR plate was treated with the following program: 95 °C for 2 min, 98°C for 30 sec, 17 cycles of (98°C for 15 sec, 64°C for 30 sec, 72°C for 2min), 72°C for 5 min then 4°C. PCR products were cleaned up using 0.8x SPRI beads and was combined into one tube for each 96–well plate. Pooled PCR product was resolved on 2% agarose gel, smear between 400 bp and 2 Kb were excised and purified using QIAquick Gel Extraction Kit (Qiagen cat. # 28706). Library concentration was determined using Qubit® dsDNA HS (High Sensitivity) Assay Kit (Invitrogen cat. # Q32851).

tag: tissue

value: frontal cortex

tag: Sex

value: Male

tag: age

value: 8 wk


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title: Pool_1165_AD010_indexed


title: Pool_1165_AD006_indexed


title: Pool_1165_AD008_indexed


title: Pool_1165_AD002_indexed


title: Pool_1164_AD008_indexed


title: Pool_1164_AD006_indexed


title: Pool_1168_AD002_indexed


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title: Pool_1163_AD006_indexed


title: Pool_1163_AD008_indexed


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title: Pool_1162_AD010_indexed


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title: Pool_1162_AD006_indexed


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title: Pool_1161_AD006_indexed


title: Pool_1162_AD002_indexed


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title: Pool_1062_AD008_indexed


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title: Pool_1058_AD008_indexed


title: Pool_1059_AD008_indexed


title: Pool_1065_AD010_indexed


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title: Pool_1049_AD008_indexed


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title: Pool_1046_AD010_indexed


title: Pool_1045_AD010_indexed


title: Pool_1046_AD008_indexed


title: Pool_1045_AD008_indexed


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title: Pool_1012_AD010_indexed


title: Pool_1006_AD006_indexed


title: Pool_1006_AD002_indexed


title: Pool_1005_AD002_indexed


title: Pool_1005_AD006_indexed


title: Pool_1004_AD002_indexed


title: Pool_1002_AD002_indexed


title: Pool_1002_AD006_indexed


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title: AS_TDP_SS4_MN_CLIP_TARDBP_input

first: Gene

last: Yeo

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

library_source: transcriptomic

data_processing:
Library strategy: eCLIP-seq
Takes output from raw files.  Run to trim off both 5’ and 3’ adapters on both reads. Command: quality-cutoff 6  -m 18  -a NNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC  -g CTTCCGATCTACAAGTT -g CTTCCGATCTTGGTCCT  -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGT  AGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT  -o /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.fastq.gz  -p /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.fastq.gz  /full/path/to/files/file_R1.C01.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.metrics
Takes output from cutadapt round 1. Run to trim off the 3’ adapters on read 2, to control for double ligation events. Command: cutadapt -f fastq --match-read-wildcards  --times 1  -e 0.1  -O 5  --quality-cutoff 6  -m 18  -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGTAGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT  -o /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.fastq.gz  -p /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.round2.fastq.gz  /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.fastq.gz  /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.fastq.gz > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.metrics
Takes output from cutadapt round 2.  Maps to human specific version of RepBase used to remove repetitive elements, helps control for spurious artifacts from rRNA (& other) repetitive reads.  Command: STAR  --runMode alignReads  --runThreadN 16  --genomeDir /path/to/RepBase_human_database_file --genomeLoad LoadAndRemove  --readFilesIn /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.fastq.gz /full/path/to/files/file_R2.C01.fastq.gz.adapterTrim.round2.fastq.gz --outSAMunmapped Within  --outFilterMultimapNmax 30  --outFilterMultimapScoreRange 1  --outFileNamePrefix /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bam --outSAMattributes All  --readFilesCommand zcat  --outStd BAM_Unsorted  --outSAMtype BAM Unsorted  --outFilterType BySJout  --outReadsUnmapped Fastx  --outFilterScoreMin 10  --outSAMattrRGline ID:foo  --alignEndsType EndToEnd > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bam
Takes output from STAR rmRep.  Maps unique reads to the human genome.  Command: STAR  --runMode alignReads  --runThreadN 16  --genomeDir  /path/to/STAR_database_file --genomeLoad LoadAndRemove  --readFilesIn  /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bamUnmapped.out.mate1  /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rep.bamUnmapped.out.mate2  --outSAMunmapped Within  --outFilterMultimapNmax 1  --outFilterMultimapScoreRange 1  --outFileNamePrefix /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam  --outSAMattributes All  --outStd BAM_Unsorted  --outSAMtype BAM Unsorted  --outFilterType BySJout  --outReadsUnmapped Fastx  --outFilterScoreMin 10  --outSAMattrRGline ID:foo  --alignEndsType EndToEnd > /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam
takes output from STAR genome mapping.  Custom random-mer-aware script for PCR duplicate removal. Command: barcode_collapse_pe.py  --bam /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.bam  --out_file /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.bam  --metrics_file /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.metrics
Takes output from barcode collapse PE.  Sorts resulting bam file for use downstream.  Command: java  -Xmx2048m  -XX:+UseParallelOldGC  -XX:ParallelGCThreads=4  -XX:GCTimeLimit=50  -XX:GCHeapFreeLimit=10  -Djava.io.tmpdir=/full/path/to/files/.queue/tmp  -cp /path/to/gatk/dist/Queue.jar  net.sf.picard.sam.SortSam  INPUT=/full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.bam  TMP_DIR=/full/path/to/files/.queue/tmp  OUTPUT=/full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam  VALIDATION_STRINGENCY=SILENT  SO=coordinate  CREATE_INDEX=true
Takes output from sortSam, makes bam index for use downstream.  Command: samtools index  /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam  /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam.bai
Takes inputs from multiple final bam files.  Merges the two technical replicates for further downstream analysis.  Command: samtools  merge  /full/path/to/files/CombinedID.merged.bam  /full/path/to/files/file_R1.C01.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam /full/path/to/files/file_R1.D08.fastq.gz.adapterTrim.round2.rmRep.rmDup.sorted.bam
Takes output from sortSam, makes bam index for use downstream.  Command: samtools  index  /full/path/to/files/CombinedID.merged.bam  /full/path/to/files/CombinedID.merged.bam.bai
Takes output from sortSam.  Only outputs the second read in each pair for use with single stranded peak caller.  This is the final bam file to perform analysis on.  Command: samtools view -hb -f 128  /full/path/to/files/CombinedID.merged.bam  >  /full/path/to/files/CombinedID.merged.r2.bam
Takes results from samtools view.  Calls peaks on those files.  Command: clipper  -b /full/path/to/files/CombinedID.merged.r2.bam  -s hg19  -o /full/path/to/files/CombinedID.merged.r2.peaks.bed  --bonferroni  --superlocal  --threshold-method binomial  --save-pickle
Genome_build: hg19
Supplementary_files_format_and_content: bed format, contains clusters of predicted RBP binding

molecule: total RNA

organism: Homo sapiens

growth_protocol: 293T cells were grown under standard conditions (DMEM + 10% FBS + Pen/Strep (100U / mL)

extract_protocol:
Cells were lysed in iCLIP lysis buffer, followed by limited digestion with RNase I (Ambion), immunoprecipitation of RBP-RNA complexes with a specific antibody of interest (Protein G sheep anti-rabbit Dynabeads), and stringent washes.
After dephosphorylation with FastAP (Thermo Fisher) and T4 PNK (NEB), a barcoded RNA adapter is ligated to the 3’ end (T4 RNA Ligase, NEB). Ligations are performed on-bead (to allow washing away unincorporated adapter) in high concentration of PEG8000, which improves ligation efficiency to > 90%. Samples are then run on standard protein gels and transferred to nitrocellulose membranes, and a region 75 kDa (~150 nt of RNA) above the protein size is isolated and proteinase K (NEB) treated to isolate RNA. RNA is reverse transcribed with AffinityScript (Agilent), and treated with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides. A second DNA adapter (containing a random-mer of 5 (N5) or 10 (N10) random bases at the 5’ end) is then ligated to the cDNA fragment 3’ end (T4 RNA Ligase, NEB), performed with high concentration of PEG8000 (to improve ligation efficiency) and DMSO (to decrease inhibition of ligation due to secondary structure). After cleanup (Dynabeads MyOne Silane, ThermoFisher), an aliquot of each sample is first subjected to qPCR (to identify the proper number of PCR cycles), and then the remainder is PCR amplified (Q5, NEB) and size selected via agarose gel electrophoresis.

treatment_protocol: While plated, cells were crosslinked with UV (254 nm, 400 mJ/cm2). Cells were then isolated with cell scrapers, pelleted at 200g, and snap frozen.

tag: cell line

tag: antibody

value: none (input)

tag: antibody manufacturer

value: input

tag: antibody catalog #

tag: antibody lot#

tag: reverse transcriptase

value: Superscript IV

tag: reverse transcriptase buffer

value: Manganese


title: Pool_1001_AD002_indexed


title: AS_TDP_SSIII_CLIP_TARDBP_input

value: Superscript III

value: Standard (magnesium)


title: AS_TDP_TGIRT_CLIP_TARDBP_input

value: TGIRT


title: TDP_SS4in3B_CLIP_AS47_TARDBP_input

value: Superscript III buffer


title: AS_TDP_SS4_CLIP_TARDBP_input


title: AS_TDP_AFFSC_CLIP_TARDBP_input

value: AffinityScript


title: AS_TDP_TGIRT_CLIP_TARDBP

value: TARDBP

value: Bethyl


title: AS_TDP_SS4_MN_CLIP_TARDBP


title: AS_TDP_SS4_CLIP_TARDBP


title: AS_TDP_AFFSC_CLIP_TARDBP


title: AS_TDP_SSIII_CLIP_TARDBP


title: TDP_SSIII_TARDBP


title: TDP_SS4in3B_TARDBP


title: TDP_TGIRT_TARDBP


title: Pool_1001_AD006_indexed


title: Pool_1003_AD006_indexed


title: 293XT_Input


title: TDP_SS4in3B_CLIP_AS47_TARDBP


title: SST_A


title: TDP_SS4_TARDBP


title: TDP_AFFSC_TARDBP


title: AS_FOX2_SSIII_CLIP_RBFOX2_input


title: AS_FOX2_SS4_MN_CLIP_RBFOX2_input


title: AS_FOX2_TGIRT_CLIP_RBFOX2_input


title: TDP_SS4_MN_TARDBP


title: AS_FOX2_SS4_CLIP_RBFOX2_input


title: AS_FOX2_AFFSC_CLIP_RBFOX2_input


title: AS_TiGER_RBFOX2


title: FOX2_SS4in3B_CLIP_-AS45_RBFOX2


title: FOX2_SS4_RBFOX2


title: FOX2_SS4in3B_RBFOX2


title: AS_FOX2_SS4_MN_CLIP_RBFOX2


title: AS_SSc4_RBFOX2


title: FOX2_TGIRT_RBFOX2


title: AS_SS4Mn_RBFOX2


title: FOX2_SSIII_RBFOX2


title: AS_FOX2_TGIRT_CLIP_RBFOX2


title: FOX2_SS4_MN_RBFOX2


title: AS_SSc3_RBFOX2


title: AS_FOX2_SS4_CLIP_RBFOX2


title: AS_SuS2_RBFOX2

value: Superscript II


title: AS_FOX2_SSIII_CLIP_RBFOX2


title: AS_M_MLV_RBFOX2

value: M-MLV


title: AS_FOX2_AFFSC_CLIP_RBFOX2


title: AS_9_Maxima_IP_RBFOX2_input

value: Maxima


title: AS_9_Maxima_IP_RBFOX2


title: AS_8_SSIII_IP_RBFOX2


title: FOX2_AFFSC_RBFOX2


title: AS_7_MaximaMn_IP_RBFOX2_input


title: AS_AMV_RBFOX2

value: AMV


title: AS_6_TGIRTMn_IP_RBFOX2_input


title: AS_8_SSIII_IP_RBFOX2_input


title: FOX2_SS4in3B_CLIP_-AS45_RBFOX2_input


title: AS_6_TGIRTMn_IP_RBFOX2


title: AS_4_SSIVMn_IP_RBFOX2_input


title: AS_4_SSIVMn_IP_RBFOX2


title: AS_7_MaximaMn_IP_RBFOX2


title: myotube_Myotonic Dystriphy type I (DM1) ctg targeting

first: Ranjan

last: Batra

state: California

address: 2880 Torrey Pines Scenic Drive

department: Cellular and Molecular Medicine

institute: University of California San Diego

data_processing:
RNA-seq data was aligned to the human hg19 genome build using Olego alignes, and alternative splicing was estimated as described below.
Quantas software as described in Charizanis et al, 2012, Neuron, was used to estimate alternative splicing. Olego aligned alignment files were used to count observed junction reads for each exon. Weighted number of exon or exon-junction fragments uniquely supporting the inclusion or skipping isoform of each cassette exon and a probability score was assigned to each isoform. A Fisher’s exact test was used to evaluate the statistical significance of splicing changes using both exon and exon-junction fragments, followed by Benjamini multiple testing correction to estimate the false discovery rate (FDR). In addition, inclusion or exclusion junction reads were used to calculate the proportional change of exon inclusion (dI). See documentation at http://zhanglab.c2b2.columbia.edu/index.php/Quantas_Documentation.
Genome_build: hg19
Supplementary_files_format_and_content: RPKM

molecule: polyA RNA

source_name: Myotubes

growth_protocol: Myoblasts were grown in complete skeletal muscle growth media SKGM-2 (Lonza) media and differentiated into myotubes in skeletal muscle basal media (SKBM-2, Lonza) supplemented with 2% Horse serum

extract_protocol:
Total RNA was isolated using TRIzol reagent using the manufacturer’s protocol (Life Technologies)
For RNA-seq, libraries were made using the Illumina polyA selection truseq library kit using different barcodes. Libraries were not strand specific.
Non-strand specific for Illumina Hiseq 4000

treatment_protocol: Myoblasts and myotubes were treated with either lentivirus containing either non-targeting (NT) or CTG-targeting (CTG) sgRNA and PIN-dCas9 (RNA targeting Cas9)  for 48 hours, selected with puromycin for 96 hours and harvested for RNA-seq library prep.

tag: treatment

value: Lentivirus transduction with PIN-dCas9 and CTG sgRNA

tag: tag

value: Puromycin selection - 4 days


title: AS_5_AffScMn_IP_RBFOX2_input

value: none (input)


title: AS_3_SSIIIMn_IP_RBFOX2_input

value: input


title: AS_3_SSIIIMn_IP_RBFOX2


title: AS_1_MMLVMn_IP_RBFOX2_input


title: AS_1_MMLVMn_IP_RBFOX2


title: myotube_Myotonic Dystriphy type I (DM1) non targeting


title: AS_2_SSIIMn_IP_RBFOX2


title: AS_2_SSIIMn_IP_RBFOX2_input


title: myoblast_Myotonic Dystriphy type I (DM1) non targeting

source_name: Myoblasts

value: Lentivirus transduction with PIN-dCas9 and non targeting sgRNA


title: myotube_Control-CTG targeting

value: Lentivirus treatment with PIN-dCas9 and non targeting sgRNA


title: myoblast_Myotonic Dystriphy type I (DM1) ctg targeting


title: myoblast_Myotonic Dystriphy type I (DM1) GFP

value: Lentivirus transduction with GFP


title: myoblast_Control-CTG targeting


title: myoblast_Control-Non targeting


title: myotube_Control-Non targeting


title: Pool_9_AD010_indexed

data_processing:
genome build: hg19
For multiplexed single nucleus methylome samples, reads were first trimmed to remove sequencing adaptors using Cutadapt 1.11 with the following parameters in paired ends mode: -f fastq -q 20 -m 62 -a AGATCGGAAGAGCACACGTCTGAAC -A AGATCGGAAGAGCGTCGTGTAGGGA. For singleplex samples, -m parameter was set to 40. For multiplexed samples, 16bp was further trimmed from both 5’- and 3’- ends of R1 and R2 reads to remove random primer index sequence and C/T tail introduced by Adaptase, with the following parameters: -f fastq -u 16 -u -16 -m 30. For singleplex samples, 10bp was trimmed from both 5’- and 3’- ends of R1 and R2 reads with the following parameters: -f fastq -u 10 -u -10 -m 30. Trimmed reads for mouse and human single nuclei were mapped to mm10 and hg19 reference genome, respectively. R1 and R2 reads were mapped separately as single-ended reads using Bismark v0.15.0 with parameter --bowtie2. --pbat option was activated for mapping R1 reads. Resulted bam files were sorted using samtools 1.3 sort, followed by removal of duplicate reads using Picard 1.141 MarkDuplicates with the option REMOVE_DUPLICATES=true. Non-clonal reads were further filtered for minimal MAPQ = 30 using samtools view with option -q30. To prevent regional mCH level estimation from being skewed by rare reads failed to be bisulfite converted, reads with read-level mCH level greater than 0.7 were excluded. The calling of unmethylated and methylated base calls were performed by call_methylated_sites of Methylpy (https://github.com/yupenghe/methylpy/) (1–3).
Supplementary_files_format_and_content: tab delimited text files of methylcytosine calls; columns in allc files are: column 1 - chromosome; column 2 - position; column 3 - strand; column 4 - class; column 5 - mC reads; column 6 - total reads; column 7 - methylated (Boolean value indicating the result of statistical test for methylated cytosines)

source_name: Human frontal cortex

value: frontal cortex

value: Male

value: 25 yr


title: UMB_4540

extract_protocol:
Whole-Genome Sequencing
Nuclei isolation from mouse and human cortical tissues was performed as described in (Lister et al., 2013) with the following modifications: Proteinase inhibitor (11836153001, Roche) and RNAse Inhibitor (30 U/ml, PRN2611 from Promega) was added to the lysis buffer and sucrose gradients. After centrifugation, nuclei were resupended in 0.5% BSA (AM2616, Ambion) PBS (Ca2+ and Mg2+ free, 14190-144 from Life Technologies,) with protein and RNAse inhibitors. Isolated nuclei from mouse and human tissues were labeled by incubation with 1:1000 dilution of AlexaFluor488 conjugated anti-NeuN antibody (MAB377X, Millipore) at 4°C for 1 hour. Nuclei isolated from CLSun1-G35-Cre line were incubated with AlexaFluor647 conjugated anti-NeuN antibody (anti-NeuN antibody MAB377 labeled using Apex Alexa Fluor 647. A10475, Life Technologies) and AlexaFluor488 conjugated anti-GFP antibody (A21311, Life Technologies). Fluorescence activated sorting of single nuclei was performed using a BD Influx sorter with an 85 µm nozzle at 22.5 PSI sheath pressure. Single nuclei were sorted into each well of a 384-well plate preloaded with 2 µl of Proteinase K digestion buffer (1 µl M-Digestion Buffer, 0.1 µl 20 µg/µl Proteinase K and 0.9 µl H2O). The alignment of receiving 384-well plate was performed by sorting sheath flow into wells of an empty plate and making adjustment based on the liquid drop position. Single cell (1 drop single) mode was selected to ensure the stringency of sorting.
Steps of library preparation prior to SPRI purification were performed in a horizontal laminar flow hood to minimize environmental DNA contamination. Bisulfite conversion of single nuclei was carried out using Zymo EZ-96 DNA Methylation-DirectTM Kit (Deep Well Format, cat. #D5023) following the product’s manual with reduced reaction volume. 384-well plate (ThermoFisher Armadillo PCR Plate cat. # AB2384) containing FACS isolated single nuclei were heated at 50°C for 20 min. 25 µl CT Conversion Reagent was added to each well followed by pipetting up-and-down to mix. Plates were treated with a following program using a thermocycler: 98°C for 8 min, 64°C for 3.5 hours and 4°C. Each well of Zymo-Spin™ I-96 Binding Plates was preloaded with 150 µl M-binding buffer. Bisulfite conversion reactions were transferred from 384-well plates to I-96 Binding Plates followed by pipetting up-and-down to mix. I-96 Binding Plates were centrifuged at 5,000g for 5 min. Wells were washed with 400 µl of M-Wash Buffer followed by centrifugation at 5,000g for 5 min. 200 µl of M-Desulphonation Buffer was added to each well and was incubated for 15 min at room temperature before removed by centrifugation at 5,000g for 5 min. Each well was then washed with 400 µl of M-Wash Buffer for two rounds. 12 µl of M-Elution Buffer was added to each well and was incubated for 5 min at room temperature. I-96 Binding Plate was placed above a 96-well PCR plate (Applied Biosystems MicroAmp® EnduraPlateTM cat. # 4483348) and was centrifuged at 5,000g for 3min. 9 µl of eluted DNA was commonly collected in each well of the PCR plate. 1 µl of 5 µM P5L-AD002-N9, P5L-AD006-N9, P5L-AD008-N9 or P5L-AD010-N9 indexed random primer was added to each well of 96-well plate followed by mixing with vortexing. All DNA oligos were purchased from Integrated DNA Technologies (IDT). Plate was heated at 95°C for 3 min to denature the sample and was immediately chilled on ice for more than 2 min. 10 µl enzyme mix containing 2 µl of Blue Buffer (Enzymatics cat. # B0110), 1 µl of  10mM dNTP (NEB cat. # N0447L), 1 µl of Klenow exo- (50U/µl, Enzymatics cat. # P7010-HC-L) and 6 µl H2O. After mixing with vortexing, plate was treated with a following program: 4°C for 5 min, ramp up to 25°C at 0.1°C/sec, 25°C for 5 min, ramp up to 37°C at 0.1°C/sec, 37°C for 60 min, 4°C. 2 µl of Exonuclease 1 (20U/µl, Enzymatics cat. # X8010L) was added to each well followed by mixing with vortexing. Plate was incubated at 37°C for 30 min and then 4°C. 17.6 µl of home-made SPRI beads was added to each well. Sample/bead mixture from four plates indexed using distinct indexed random primers were combined followed by pipetting up-and-down to mix. Sample/bead mixture was incubated at room temperature for 5 min before placed on a 96-well magnetic separator. Supernatant was removed from each well followed by three rounds of washing with 180 µl of 80% ethanol. After air drying beads at room temperature, 10 µl M-Elution buffer was added to each well to fully resuspend the beads. Eluted sample was transferred to a new 96-well PCR plate. PCR plate was heated at 95°C for 3 min to denature the sample and was immediately chilled on ice for more than 2 min. 10.5 µl Adaptase (Swift Biosciences) mix (2 µl Buffer G1, 2 µl Reagent G2, 1.25 µl Reagent G3, 0.5 µl Enzyme G5, 0.5 µl Enzyme G6 and 4.25 µl M-Elution buffer) was added into each well followed by mixing with vortexing. Plates was incubated at 37°C at 30 min and then 4°C. 30 µl PCR mix (25 µl KAPA HiFi HotStart ReadyMix, KAPA BIOSYSTEMS, cat. # KK2602, 1 µl 30 µM P5 indexing primer and 5 µl 10 µM P7 indexing primer) was added into each well followed by mixing with vortexing. PCR plate was treated with the following program: 95 °C for 2 min, 98°C for 30 sec, 17 cycles of (98°C for 15 sec, 64°C for 30 sec, 72°C for 2min), 72°C for 5 min then 4°C. PCR products were cleaned up using 0.8x SPRI beads and was combined into one tube for each 96–well plate. Pooled PCR product was resolved on 2% agarose gel, smear between 400 bp and 2 Kb were excised and purified using QIAquick Gel Extraction Kit (Qiagen cat. # 28706). Library concentration was determined using Qubit® dsDNA HS (High Sensitivity) Assay Kit (Invitrogen cat. # Q32851).


title: Pool_9_AD008_indexed


title: Pool_99_AD010_indexed


title: Pool_99_AD008_indexed


title: AS_5_AffScMn_IP_RBFOX2

value: AffinityScript

value: Manganese


title: Pool_999_AD010_indexed


title: Pool_999_AD008_indexed


title: Pool_998_AD008_indexed


title: Pool_998_AD010_indexed


title: Pool_996_AD008_indexed


title: Pool_995_AD008_indexed


title: Pool_997_AD008_indexed


title: Pool_995_AD010_indexed


title: Pool_994_AD008_indexed


title: Pool_994_AD010_indexed


title: Pool_997_AD010_indexed


title: Pool_992_AD010_indexed


title: Pool_991_AD010_indexed


title: Pool_990_AD010_indexed


title: Pool_993_AD010_indexed


title: Pool_991_AD008_indexed


title: Pool_990_AD008_indexed


title: Pool_992_AD008_indexed


title: Pool_993_AD008_indexed


title: Pool_988_AD010_indexed


title: Pool_989_AD010_indexed


title: Pool_989_AD008_indexed


title: Pool_98_AD010_indexed


title: Pool_988_AD008_indexed


title: Pool_986_AD008_indexed


title: Pool_985_AD008_indexed


title: Pool_987_AD010_indexed


title: Pool_987_AD008_indexed


title: Pool_986_AD010_indexed


title: Pool_984_AD010_indexed


title: Pool_984_AD008_indexed


title: Pool_983_AD008_indexed


title: Pool_981_AD010_indexed


title: Pool_981_AD008_indexed


title: Pool_980_AD008_indexed