title: Human induced pluripotent stem cell line established from PD patient 3 clone 1 [small RNA-seq]

value: PD patient 3


title: Human induced pluripotent stem cell line established from PD patient 2 clone 2 [small RNA-seq]

status: Public on Oct 08 2018

type: SRA

city: Regensburg

first: Markus

last: Schulze

address: Franz-Josef-Strauss Allee 11

department: Neuropathology

country: Germany

institute: University Hospital Regensburg

library_source: transcriptomic

data_processing:
Image analysis and base calling resulted in .bcl files, which were converted into .fastq files by the CASAVA1.8.2 software
Fastq files were imported to the commercially available Genomatix mining station with the data import and validation tool ver. 2.2.2
Fastq files were mapped against small RNA libraries (mature miRNAs and piRNAs, annotation based on ElDorado 6-2015) allowing one mismatch with the commercially available Genomatix mapper ver. 3.7.6.3
Count tables were created for each small RNA type (piRNA, mature miRNA) with the small RNA workflow available in the Genomatix Genome Analyzer v3.51106 based on the annotation ElDorado 6-2015. Normalized expression values were calculated by dividing the counts per small RNA by the sum of all small RNA counts in a sample.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab delimited file with counts and normalized expression values, columns are column1: miRNA_Genomatix_ID column2: miRNA_Name column3: miRNA_Count column4:  miRNA_NE column5: empty column6: piRNA_Genomatix_ID column 7: piRNA_Name column8:piRNA_Count  column9: piRNA_NE

molecule: total RNA

organism: Homo sapiens

extract_protocol:
Small RNA libraries were prepared from 1 µg total RNA containing small RNAs with the TrueSeq Small RNA kit (Illumina) according to the manufacturer´s instructions. Briefly, a 3´adapter was added to the RNA, denaturation was performed for two minutes at 70°C and afterwards the mixture was put on ice immediately. Then, the adapter was ligated with a T4 RNA Ligase2 deletion mutant (epicentre) for 1h at 28°C. Then the reaction was stopped with stop solution for 15 minutes at 28°C, the previously denatured 5´ adapter was added together with ATP and T4 DNA Ligase and ligated to the RNA for 1h at 28°C. After that, cDNA synthesis was performed with super script II and Illumina supplied dNTPs for 1h at 50°C. Afterwards, the cDNA was amplified and indexed with the primers and PCR mix supplied in the kit (eleven cycles of denaturation at 98°C for 10 seconds,annealing 60°C for 30 seconds and extension at 72°C for 15 seconds with a final extension at 72 for 10 minutes) and size selection was performed on 5% TBE acrylamide gels (Bio-Rad, munich, Germany). Here, the region marked by Illumina´s custom ladder between 145 bp and 160 bp were cut out and pooled for sequencing. The gel was homogenized by centrifugation at 20000 xg through a gel breaker tube (Bio-Cat) and 300 µl ultrapure H2O were added to elute the DNA O/N in a DNA LoBind tube (Eppendorf). The following day, the gel debris was separated from the water by centrifugation for 10 seconds at 600 xg through a 5µm filter tube (Bio-Cat). Then, 2 µl glycogen (CALBIOCHEM), 30 µl sodium acetate (Thermo-Fisher Scientific), 2 µl 0.1x pellet paint and 975 µl prechilled 100% ethanol were added and the mixture was incubated for 20 minutes at -80°C. Then, the library was pelleted by centrifugation for 20 minutes at 4°C and 20000 xg, washed with 70% ethanol and recentrifuged for 5 minutes. Finally, the ethanol was removed, the pellet dried for five minutes at 37°C and resuspended in 15 µl ultrapure water.
High throughput sequencing of small RNA

tag: patient status/id

value: PD patient 2

tag: type

value: induced pluripotent stem cell

tag: molecule subtype

value: total RNA containing small RNA


title: Human induced pluripotent stem cell line established from PD patient 3 clone 2 [small RNA-seq]


title: Human induced pluripotent stem cell line established from control patient 2 clone 3 [small RNA-seq]

value: control patient 2


title: Differentiated neurons established from PD patient 4 clone 1 [small RNA-seq]

value: PD patient 4

value: differentiated midbrain neuron


title: Human induced pluripotent stem cell line established from PD patient 2 clone 1 [small RNA-seq]


title: Human induced pluripotent stem cell line established from control patient 4 clone 2 [small RNA-seq]

value: control patient 4


title: Human induced pluripotent stem cell line established from control patient 5 clone 2 [small RNA-seq]

value: control patient 5


title: Human induced pluripotent stem cell line established from PD patient 1 clone 2 [small RNA-seq]

value: PD patient 1


title: Human induced pluripotent stem cell line established from control patient 3 clone 1 [small RNA-seq]

value: control patient 3


title: Human induced pluripotent stem cell line established from control patient 1 clone 2 [small RNA-seq]

value: control patient 1


title: Human induced pluripotent stem cell line established from control patient 2 clone 1 [small RNA-seq]


title: Human induced pluripotent stem cell line established from control patient 3 clone 2 [small RNA-seq]


title: Differentiated neurons established from control patient 1 clone 2 [small RNA-seq]


title: Human induced pluripotent stem cell line established from control patient 1 clone 1 [small RNA-seq]


title: Human dermal fibroblasts established from PD patient 9 [small RNA-seq]

value: PD patient 9

value: human dermal fibroblast


title: Human dermal fibroblasts established from PD patient 8 [small RNA-seq]

value: PD patient 8


title: Human dermal fibroblasts established from PD patient 7 [small RNA-seq]

value: PD patient 7


title: Human induced pluripotent stem cell line established from control patient 3 clone 3 [small RNA-seq]


title: Human induced pluripotent stem cell line established from control patient 4 clone 1 [small RNA-seq]


title: Human dermal fibroblasts established from PD patient 6 [small RNA-seq]

value: PD patient 6


title: Human dermal fibroblasts established from PD patient 4 [small RNA-seq]


title: Human induced pluripotent stem cell line established from control patient 2 clone 2 [small RNA-seq]


title: Human dermal fibroblasts established from PD patient 5 [small RNA-seq]

value: PD patient 5


title: Human dermal fibroblasts established from control patient 5 [small RNA-seq]


title: Human dermal fibroblasts established from PD patient 1 [small RNA-seq]


title: Differentiated neurons established from PD patient 5 clone 1 [RRBS-seq]

library_source: genomic

data_processing:
Image analysis and base calling resulted in .bcl files, which were converted into .fastq files by the CASAVA1.8.2 software
fastq files were trimmed with TRIM Galore v0.4.2 in the rrbs mode
Trimmed fastq-files were mapped with BISMARK v0.16.3 57 to the human genome GRCh38.84 as obtained from www.ensembl.org
Methylation values were extracted with the BISMARK methylation extractor again removing 2 bp at the five prime end of the reads to remove the methylation bias in  the untrimmed reads stemming from the end repair procedure. In addition, only CpG dinucleotides with a coverage of at least 5x were retained.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab delimited file with methylation values, columns are column1: Chromosome column2: Start_coordinate column3: End_coordinate column4: Methylation_percentage column5: Count_methylated column6: Count_unmethylated

molecule: genomic DNA

extract_protocol:
Digestion was performed with 2.5 µg genomic DNA in buffer 4 (NEB) and 400 units MSPI O/N at 37°C. Afterwards, the DNA was purified from this reaction with the PCR purification Kit (Qiagen) according to the manufacturer´s instructions. Briefly, 5 volumes buffer PB were added to the MSPI digest, applied to a spin column provided in the kit and centrifugation was performed for 1 minute at 9500 x g. Then, the column was washed with 750 µl buffer PE, dried by centrifugation for 1 minute at 9500 x g and the DNA was eluted with 30 µl buffer EB. Library preparation was then performed with the NEXTflex™ Bisulfite Library Prep Kit (BIOO Scientific) according to the manufacturer´s instructions with some modifications. Briefly, end repair was performed with 500 ng digested, purified DNA in end repair buffer mix and end repair enzyme mix in a total volume of 50 µl. The reaction was incubated at 22°C for 30 minutes and then cleaned up with the MinElute® PCR Cleanup Kit. Then, 16.5 µl of the eluate were mixed with 4.5 µl of adenylation mix and the reaction was incubated for 30 minutes at 37 °C. Afterwards, 31.5 µl ligation mix and 2.5 µl of individual adapters (diluted 1:2) were added, and adapter ligation was performed for 15´ at 22°C. Afterwards, the DNA was cleaned with AMPure XP beads and size selection for fragments form 175 to 400 bp  was performed with a gel purification step. The libraries were separated on a 2% low melt agarose gel (Sigma-Aldrich), the cut out gel fragments were dissolved for 10 minutes at RT in DNA binding buffer and 150 µl ethanol were added. Then, the solution was applied to a clean up spin column and centrifuged at 18500 xg until the complete volume was processed. Afterwards, the column was washed twice with DNA wash buffer, dried by centrifugation and the DNA was eluted with column elution buffer. Then, bisulfite conversion of the DNA was performed with the EZ Methylation Gold Kit (Zymo Research) according to the manufacturer´s instructions. Briefly, 130 µl conversion reagent were added to 20 µl purified DNA. The reaction was incubated for 10 minutes at  98°C and for 2.5 hours at 64°C. Then, the samples were loaded on spin columns containing 600 µl M-Binding buffer and mixed by inverting. The DNA was bound to the column by centrifugation for 30 seconds at 18620 x g. Then, the column was wahed with 100 µl wash buffer, and 200 µl desulphonation buffer were added. The desulphonation buffer was incubated for 17 minutes at RT, and then removed by centrifugation. The column was washed twice with 200 µl wash buffer, and dried by centrifugation for 10 seconds. Finally, 17 µl elution buffer were added to the column, incubated for 1 minute and  the DNA was eluted by centrifugation. Afterwards, PCR amplification of the bisulfite converted libraries was performed with PfuCx Hot Start (Agilent). 15 µl DNA were amplified with the NEXTflex™ Primer Mix. Cycling conditions were: Initial denaturation for five minutes at 95°C. Then 18 cycles of 95°C for 30 seconds, 65°C for 30 seconds and 72°C for 45 seconds. Afterwards a final extension at 72°C for 7 minutes. The libraries were purified again using AMPure  XP beads (Beckmann Coulter).
High throughput sequencing of MSPI digested, size selected and  bisulfite converted genomic DNA

tag: cell type


title: Human dermal fibroblasts established from PD patient 2 [small RNA-seq]

value: PD patient 2


title: Human dermal fibroblasts established from control patient 3 [small RNA-seq]


title: Differentiated neurons established from PD patient 4 clone 1 [RRBS-seq]


title: Human dermal fibroblasts established from control patient 4 [small RNA-seq]


title: Human dermal fibroblasts established from control patient 6 [small RNA-seq]

value: control patient 6


title: Differentiated neurons established from PD patient 2 clone 2 [RRBS-seq]


title: Human dermal fibroblasts established from control patient 2 [small RNA-seq]

value: total RNA containing small RNA


title: Differentiated neurons established from PD patient 1 clone 1 [RRBS-seq]


title: Differentiated neurons established from control patient 5 clone 1 [RRBS-seq]


title: Differentiated neurons established from control patient 1 clone 2 [RRBS-seq]


title: Human dermal fibroblasts established from control patient 1 [small RNA-seq]


title: Differentiated neurons established from control patient 2 clone 2 [RRBS-seq]

value: control patient 2


title: Differentiated neurons established from control patient 3 clone 1 [RRBS-seq]


title: Differentiated neurons established from control patient 4 clone 1 [RRBS-seq]


title: Differentiated neurons established from PD patient 3 clone 1 [RRBS-seq]

value: PD patient 3


title: Embryonic stem cell line H9 [RRBS-seq]

source_name: Embryonic stem cell line H9

tag: cell line

value: embryonic stem cell


title: Human induced pluripotent stem cell line established from PD patient 5 clone 3 [RRBS-seq]

value: induced pluripotent stem cell


title: Human induced pluripotent stem cell line established from PD patient 5 clone 1 [RRBS-seq]


title: Embryonic stem cell line HUES6 [RRBS-seq]

source_name: Embryonic stem cell line HUES6


title: Human induced pluripotent stem cell line established from PD patient 8 clone 2 [RRBS-seq]


title: Human induced pluripotent stem cell line established from PD patient 4 clone 3 [RRBS-seq]

value: PD patient 4


title: Human induced pluripotent stem cell line established from PD patient 4 clone 2 [RRBS-seq]


title: Human dermal fibroblasts established from PD patient 3 [small RNA-seq]


title: Human induced pluripotent stem cell line established from PD patient 4 clone 1 [RRBS-seq]


title: Human induced pluripotent stem cell line established from PD patient 3 clone 2 [RRBS-seq]


title: Human induced pluripotent stem cell line established from PD patient 2 clone 2 [RRBS-seq]


title: Human induced pluripotent stem cell line established from PD patient 3 clone 1 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 5 clone 2 [RRBS-seq]

value: control patient 5


title: Human induced pluripotent stem cell line established from PD patient 1 clone 1 [RRBS-seq]

value: PD patient 1


title: Human induced pluripotent stem cell line established from control patient 5 clone 1 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 4 clone 2 [RRBS-seq]

value: control patient 4


title: Human induced pluripotent stem cell line established from control patient 3 clone 1 [RRBS-seq]

value: control patient 3


title: Human induced pluripotent stem cell line established from PD patient 1 clone 2 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 4 clone 1 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 6 clone 1 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 3 clone 3 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 2 clone 3 [RRBS-seq]


title: Human induced pluripotent stem cell line established from PD patient 2 clone 1 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 2 clone 2 [RRBS-seq]


title: Human dermal fibroblasts established from PD patient 9 [RRBS-seq]

value: PD patient 9

value: human dermal fibroblast


title: Human dermal fibroblasts established from PD patient 8 [RRBS-seq]

value: PD patient 8


title: Human induced pluripotent stem cell line established from control patient 1 clone 2 [RRBS-seq]

value: control patient 1


title: Human induced pluripotent stem cell line established from control patient 1 clone 1 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 2 clone 1 [RRBS-seq]


title: Human induced pluripotent stem cell line established from control patient 6 clone 2 [RRBS-seq]


title: Human dermal fibroblasts established from PD patient 7 [RRBS-seq]

value: PD patient 7


title: Human induced pluripotent stem cell line established from control patient 3 clone 2 [RRBS-seq]


title: Human dermal fibroblasts established from PD patient 6 [RRBS-seq]

value: PD patient 6


title: Human dermal fibroblasts established from PD patient 5 [RRBS-seq]

value: PD patient 5


title: Human dermal fibroblasts established from PD patient 3 [RRBS-seq]


title: Human dermal fibroblasts established from PD patient 2 [RRBS-seq]

value: PD patient 2


title: Human dermal fibroblasts established from PD patient 4 [RRBS-seq]


title: Human dermal fibroblasts established from PD patient 1 [RRBS-seq]


title: Human dermal fibroblasts established from control patient 6 [RRBS-seq]

value: control patient 6


title: Differentiated neurons established from control patient 3 clone 3

data_processing:
Image analysis and base calling resulted in .bcl files, which were converted into .fastq files by the CASAVA1.8.2 software
Fastq files were imported to the commercially available Genomatix mining station with the data import and validation tool ver. 2.2.2
Reads were mapped to the human genome version GRCh38 with the commercially available Genomatix mapper ver. 3.7.6.3 allowing one mismatch
Count tables as well as RPKM values were created by the Genomatix Genome Analyzer v3.51106 based on the annotation ElDorado 6-2015. Reads were counted locus-based, i.e. for unions of exons of genes.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab delimited file with counts and RPKM values, columns are column1: Gene_Symbol column2: Chromosome column3: Start_coordinate column 4: End_coordinate 5: Strand 6: Count C column7: RPKM

extract_protocol:
Libraries for next-generation sequencing were prepared from total RNA with the TruSeq® RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer´s instructions. Poly-A RNA was purified from the total RNA preparation with magnetic oligo-dT beads. The RNA-bead mixture was incubated at 65°C for five minutes to denature the RNA. Then, the mixture was incubated for five minutes at RT to allow the RNA to bind to the beads. Afterwards, the beads were washed with bead washing buffer and resuspended in elution buffer for two minutes at 80°C. Bead binding buffer was added to allow rebinding of the eluted RNA. The beads were washed with bead washing buffer and resuspended in elute, prime, fragment mix. The RNA was eluted at 94°C for eight minutes, and first strand cDNA synthesis was performed with SuperScript II (Thermo Fisher Scientific, Waltham, MA, USA) in first strand mix supplied by Illumina. Second strand synthesis was performed with the second strand mix for one hour at 16°C and the resulting double stranded cDNA was purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). Then, end repair was performed for 30 minutes at 30°C with the provided end repair mix. The end repaired DNA was again cleaned up with AMPure XP beads. A-tailing was performed with the A-tailing mix at 37°C for 30 minutes followed by 70°C for five minutes. Indexed adapters were ligated to the end-repaired A-tailed cDNA at 30°C for ten minutes. Next, stop ligation buffer was added, and the libraries were cleaned up with AMPure XP beads. PCR amplification was performed with the provided PCR reagents and the following cycling conditions: Denaturation at 98°C for 30 seconds and then 15 cycles of1) 98°C for 10 seconds, 2)60°C for 30 seconds and 3)72°C for 30 seconds. Afterwards, a final extension at 72°C for five minutes was performed and the amplified libraries were purified again with AMPure XP beads.
High throughput sequencing of poly-A RNA

value: differentiated midbrain neuron


title: Differentiated neurons established from PD patient 4 clone 1


title: Human dermal fibroblasts established from control patient 2 [RRBS-seq]

value: control patient 2


title: Human dermal fibroblasts established from control patient 1 [RRBS-seq]


title: Differentiated neurons established from control patient 2 clone 3


title: Human dermal fibroblasts established from control patient 4 [RRBS-seq]


title: Differentiated neurons established from PD patient 6 clone 1


title: Differentiated neurons established from control patient 5 clone 1


title: Differentiated neurons established from PD patient 3 clone 2

value: PD patient 3


title: Differentiated neurons established from PD patient 5 clone 1


title: Human dermal fibroblasts established from control patient 3 [RRBS-seq]


title: Differentiated neurons established from PD patient 2 clone 2


title: Differentiated neurons established from PD patient 1 clone 1


title: Differentiated neurons established from control patient 1 clone 1


title: Differentiated neurons established from control patient 3 clone 1


title: Embryonic stem cell line HUES6

value: embryonic stem cell


title: Embryonic stem cell line H9


title: Differentiated neurons established from control patient 1 clone 2


title: Human induced pluripotent stem cell line established from PD patient 9 clone 1

value: induced pluripotent stem cell


title: Differentiated neurons established from control patient 2 clone 2


title: Human induced pluripotent stem cell line established from PD patient 8 clone 2


title: Human dermal fibroblasts established from control patient 5 [RRBS-seq]

value: control patient 5


title: Human induced pluripotent stem cell line established from PD patient 4 clone 3

value: PD patient 4


title: Human induced pluripotent stem cell line established from PD patient 6 clone 1


title: Human induced pluripotent stem cell line established from PD patient 5 clone 3


title: Human induced pluripotent stem cell line established from PD patient 5 clone 1


title: Human induced pluripotent stem cell line established from PD patient 4 clone 1


title: Differentiated neurons established from PD patient 3 clone 1


title: Human induced pluripotent stem cell line established from PD patient 3 clone 2


title: Human induced pluripotent stem cell line established from PD patient 3 clone 1


title: Human induced pluripotent stem cell line established from PD patient 2 clone 2


title: Human induced pluripotent stem cell line established from PD patient 2 clone 1


title: Human induced pluripotent stem cell line established from PD patient 9 clone 2


title: Differentiated neurons established from control patient 4 clone 1

value: control patient 4


title: Human induced pluripotent stem cell line established from control patient 5 clone 1


title: Human induced pluripotent stem cell line established from PD patient 1 clone 2

value: PD patient 1


title: Human induced pluripotent stem cell line established from control patient 6 clone 2


title: Human induced pluripotent stem cell line established from control patient 5 clone 2


title: Human induced pluripotent stem cell line established from PD patient 1 clone 3


title: Human induced pluripotent stem cell line established from control patient 5 clone 3


title: Human induced pluripotent stem cell line established from PD patient 1 clone 1


title: Human induced pluripotent stem cell line established from control patient 4 clone 1


title: Human induced pluripotent stem cell line established from control patient 4 clone 2


title: Human induced pluripotent stem cell line established from control patient 3 clone 3

value: control patient 3


title: Human induced pluripotent stem cell line established from control patient 3 clone 1


title: Human induced pluripotent stem cell line established from control patient 3 clone 2


title: Human induced pluripotent stem cell line established from control patient 2 clone 2


title: Human induced pluripotent stem cell line established from control patient 2 clone 1


title: Human induced pluripotent stem cell line established from control patient 2 clone 3


title: Human induced pluripotent stem cell line established from control patient 1 clone 1


title: Human dermal fibroblasts established from PD patient 7

value: human dermal fibroblast


title: Human induced pluripotent stem cell line established from control patient 1 clone 2

value: control patient 1


title: Human induced pluripotent stem cell line established from PD patient 4 clone 2


title: Human dermal fibroblasts established from PD patient 9

value: PD patient 9


title: Human dermal fibroblasts established from PD patient 8

value: PD patient 8


title: Human dermal fibroblasts established from PD patient 6

value: PD patient 6


title: Human induced pluripotent stem cell line established from control patient 6 clone 1


title: Human dermal fibroblasts established from PD patient 4


title: Human dermal fibroblasts established from PD patient 1


title: Human dermal fibroblasts established from PD patient 2

value: PD patient 2


title: Human dermal fibroblasts established from PD patient 5

value: PD patient 5


title: Human dermal fibroblasts established from control patient 1


title: Human dermal fibroblasts established from control patient 4


title: Human dermal fibroblasts established from control patient 5


title: Cingulate gyrus tissue from tissue donor PD 8

extract_protocol:
Libraries were prepared with the TrueSeq RNA Access Kit (Illumina), which is suitable for degraded RNA. First, first strand cDNA was synthetized with the Elute, Prime, Fragment High Mix and super script II in First Strand Master Mix (25°C for 10 minutes, 42°C for 15 minutes and 70°C for 15 minutes). Then, 20 µl Second Strand Marking Master Mix were added and the second strand was synthetized for 1h at 16°C. The cDNA was cleaned up with AMPure XP beads and A-tailing was performed with A-tailing mix (37°C for 30 minutes followed by 5 minutes at 70°C). Following this, adapter ligation was performed with Ligation Mix for 10 minutes at 30°C after which the reaction was stopped with Stop Ligation Buffer. The libraries were cleaned up with AMPure XP beads and amplified with PCR Master Mix and PCR Primer Cocktail (98°C for 30 seconds, 15 cycles of 98°C for 10 seconds, 60°C for 30 seconds, 72°C for 30 seconds and 72°C for 5 minutes). Following a further clean up with AMPure XP beads, the libraries were quantitated with a Bio-Analyser and 4 libraries were pooled at equal concentrations (200 ng each) for exome capture, which was repeated twice. For exome capture, Coding Exome Oligos were added to the pooled libraries together with Capture Target Buffer 3 and incubated for 95°C for 10 minutes and 18 cycles of one minute incubations, starting at 94°C, then decreasing 2°C per cycle with a final incubation for 58°C for 90 minutes. Immediately afterwards, the hybridized probes were captured with streptavidin magnetic beads for 25 minutes at RT and washed twice with Enrichment Wash Solution (incubation at 50°C for 20 minutes). Finally an Elution Premix was prepared with Enrichment Elution Buffer 1 and NaOH. The streptavidin beads were resuspended in this Elution Premix, incubated for two minutes at RT, the supernatant was separated from the beads on a magnetic stand  and Elute Target Buffer 2 was added to the supernatant. After the second exome capture, the libraries were cleaned up with AMPure XP beads. Finally, a second enrichment was performed with the Enhanced PCR Mix (98°C for 30 seconds followed by 10 cycles of 98°C for 10 seconds, 60°C for 30 seconds and 72°C for 30 seconds, with a final extension at 72°C for 5 minutes) and the amplified libraries were purified again with AMPure XP beads.
High throughput sequencing of RNA (coding exome)

value: Parkinson´s disease patient 8

tag: tissue

value: brain

tag: tissue subtype

value: cingulate gyrus


title: Cingulate gyrus tissue from tissue donor PD 3

value: Parkinson´s disease patient 3


title: Cingulate gyrus tissue from tissue donor PD 7

value: Parkinson´s disease patient 7


title: Cingulate gyrus tissue from tissue donor PD 6

value: Parkinson´s disease patient 6


title: Cingulate gyrus tissue from tissue donor PD 4

value: Parkinson´s disease patient 4


title: Human dermal fibroblasts established from control patient 2

value: control patient 2


title: Cingulate gyrus tissue from tissue donor control 3

value: control donor 3


title: Cingulate gyrus tissue from tissue donor PD 1

value: Parkinson´s disease patient 1


title: Human dermal fibroblasts established from control patient 6

value: control patient 6

value: human dermal fibroblast


title: Human dermal fibroblasts established from PD patient 3

value: PD patient 3


title: Cingulate gyrus tissue from tissue donor PD 2

value: Parkinson´s disease patient 2


title: Cingulate gyrus tissue from tissue donor control 8

value: control donor 8


title: Cingulate gyrus tissue from tissue donor control 6

value: control donor 6


title: Cingulate gyrus tissue from tissue donor control 7

value: control donor 7


title: Human dermal fibroblasts established from control patient 3

value: control patient 3


title: Cingulate gyrus tissue from tissue donor control 4

value: control donor 4

value: brain


title: ovule_3.nanoPARE

city: Wageningen

first: Michael

middle: D

last: Nodine

state: Gelderland

address: Radix West

department: Plant Sciences

country: Netherlands

institute: Wageningen University & Research

description:
Single-end 50 base read nanoPARE data from third biological replicate of ovules from stage 13 Col-0 flowers
Sample 48

data_processing:
Library strategy: nanoPARE
mRNA-Seq: Sequences in FASTQ files were trimmed with cutadapt, filtered for low-complexity and then aligned to the Arabopsis thaliana Col-0 genome (TAIR10) with STAR with the following settings: --runMode alignReads; --alignIntronMax 10000; --alignMatesGapMax 11000; --alignSJDBoverhangMin 1; --alignSJoverhangMin 10; --outSJfilterOverhangMin -1 10 10 10; --outSJfilterCountTotalMin -1 2 2 2; --outSJfilterCountUniqueMin -1 2 2 2; --outFilterType BySJout; --outFilterMismatchNmax 2; --outFilterMismatchNoverLmax .05; --twopassMode Basic; --outFilterIntronMotifs RemoveNoncanonicalUnannotated; --outFilterMultimapNmax 100; --outSAMmultNmax 100; --outSAMattributes NH HI AS nM NM MD jM jI XS; --outSAMprimaryFlag AllBestScore; --outSAMtype BAM Unsorted; --alignEndsType EndToEnd. Aligned BAM files were used to calculate 5' end read depth for each position in the genome using the Python script readmapIO, which employs a "rich-get-richer" algorithm to assign reads that map to multiple locations, and outputs bedgraph files.
nanoPARE: Sequences in FASTQ files were trimmed with cutadapt, filtered for low-complexity and then aligned to the Arabopsis thaliana Col-0 genome (TAIR10) with STAR with the following settings: --runMode alignReads; --alignIntronMax 10000; --alignMatesGapMax 11000; --alignSJDBoverhangMin 1; --alignSJoverhangMin 10; --outSJfilterOverhangMin -1 10 10 10; --outSJfilterCountTotalMin -1 2 2 2; --outSJfilterCountUniqueMin -1 2 2 2; --outFilterType BySJout; --outFilterMismatchNmax 2; --outFilterMismatchNoverLmax .05; --twopassMode Basic; --outFilterIntronMotifs RemoveNoncanonicalUnannotated; --outFilterMultimapNmax 100; --outSAMmultNmax 100; --outSAMattributes NH HI AS nM NM MD jM jI XS; --outSAMprimaryFlag AllBestScore; --outSAMtype BAM Unsorted; --alignEndsType Local; --outFilterMatchNminOverLread 0.9. Aligned BAM files were used to calculate 5' end read depth for each position in the genome using the Python script readmapIO, which employs a "rich-get-richer" algorithm to assign reads that map to multiple locations, and outputs bedgraph files, which also include all nucleotides softclipped from the 5' ends of reads (upstream untemplated nucleotides (uuNs). Nucleotide-specific bias correction was applied as in Wange et al. (2017) BMC Bioinformatics with the following modifications. To apply the method to RNA-Seq data rather than DNA-Seq data, only the presence or absence of reads at a position was considered, not read abundance. K-mer frequencies of length 3 were calculated for reads mapping to the coding regions of TAIR10 genes, and bias correction against this k-mer matrix was applied to all reads.Discrete 5P features were identified from these alignments via subtractive kernel density estimation of the a sample's nanoPARE and mRNA-Seq libraries. Each feature was classified as capped if ≥10% of all reads from the sample contain uuG or classified as noncapped otherwise. Bedgraph files of 5P read positions relative to the start site of the dominant isoform of each gene in the reference annotation were prepared and capped features were masked for sRNA-mediated cleavage site analyses.
Genome_build: TAIR10
Supplementary_files_format_and_content: Bedgraph files contain tab-separated values of chromosomes or dominant transcripts (column 1), start (column 2) and stop (column 3) coordinates of 5' end of alignments and number of aligned ends weighted by their number of alignments.

organism: Arabidopsis thaliana

source_name: ovules from stage 13 flowers

growth_protocol: Controlled growth chamber at 20˚C-22˚C with a 16-h light/8-h dark cycle. Plants were four weeks old when tissue was harvested.

extract_protocol:
TRIzol reagent (Thermo Fisher Scientific)
For Samples 1-6, mRNA-Seq libraries were generated according to the Smart-Seq2 protocol (Picelli et al. 2013. Nature Methods). For Samples 7-48, nanoPARE libraries were generated as detailed in the associated article.

treatment_protocol: RNA was optionally incubated with XRN-1 exoribonuclease (NEB) for one hour at 37˚ C prior to library construction

value: ovules from stage 13 flowers

tag: ecotype

tag: genotype

value: wild type

tag: rna treatment prior to library construction

tag: amount of total rna used for library construction (µg)


title: Cingulate gyrus tissue from tissue donor control 1

value: control donor 1

value: cingulate gyrus


title: Cingulate gyrus tissue from tissue donor control 2

value: control donor 2


title: stigma_1.nanoPARE

description:
Single-end 50 base read nanoPARE data from first biological replicate of stigmas from stage 13 Col-0 flowers
Sample 43

source_name: stigmas from stage 13 flowers

value: stigmas from stage 13 flowers


title: Cingulate gyrus tissue from tissue donor control 5

value: control donor 5


title: stigma_3.nanoPARE

description:
Single-end 50 base read nanoPARE data from third biological replicate of stigmas from stage 13 Col-0 flowers
Sample 45


title: stigma_2.nanoPARE

description:
Single-end 50 base read nanoPARE data from second biological replicate of stigmas from stage 13 Col-0 flowers
Sample 44


title: petal_1.nanoPARE

description:
Single-end 50 base read nanoPARE data from first biological replicate of petals from stage 13 Col-0 flowers
Sample 37

source_name: petals from stage 13 flowers

value: petals from stage 13 flowers


title: petal_2.nanoPARE

description:
Single-end 50 base read nanoPARE data from second biological replicate of petals from stage 13 Col-0 flowers
Sample 38


title: ovule_1.nanoPARE

description:
Single-end 50 base read nanoPARE data from first biological replicate of ovules from stage 13 Col-0 flowers
Sample 46


title: anther_3.nanoPARE

description:
Single-end 50 base read nanoPARE data from third biological replicate of anthers from tage 13 Col-0 flowers
Sample 42

source_name: anthers from stage 13 flowers

value: anthers from stage 13 flowers


title: Cingulate gyrus tissue from tissue donor PD 5

value: Parkinson´s disease patient 5


title: sepal_2.nanoPARE

description:
Single-end 50 base read nanoPARE data from second biological replicate of sepals from stage 13 Col-0 flowers
Sample 35

source_name: sepals from stage 13 flowers

value: sepals from stage 13 flowers


title: anther_1.nanoPARE

description:
Single-end 50 base read nanoPARE data from first biological replicate of anthers from stage 13 Col-0 flowers
Sample 40


title: petal_3.nanoPARE

description:
Single-end 50 base read nanoPARE data from third biological replicate of petals from stage 13 Col-0 flowers
Sample 39


title: anther_2.nanoPARE

description:
Single-end 50 base read nanoPARE data from second biological replicate of anthers from stage 13 Col-0 flowers
Sample 41


title: ovule_2.nanoPARE

description:
Single-end 50 base read nanoPARE data from second biological replicate of ovules from stage 13 Col-0 flowers
Sample 47


title: sepal_1.nanoPARE

description:
Single-end 50 base read nanoPARE data from first biological replicate of sepals from 13 Col-0 flowers
Sample 34


title: sepal_3.nanoPARE

description:
Single-end 50 base read nanoPARE data from third biological replicate of sepals from stage 13 Col-0 flowers
Sample 36


title: flower_3.nanoPARE

description:
Single-end 50 base read nanoPARE data from third biological replicate of whole stage 13 Col-0 flowers
Sample 33

source_name: whole stage 13 flowers

value: whole stage 13 flowers


title: 10pg_fb_3.nanoPARE

description: Single-end 50 base read nanoPARE data from third biological replicate of Col-0 flowers (10 pg)

data_processing:
Sequences in FASTQ files were trimmed with cutadapt, filtered for low-complexity and then aligned to the Arabopsis thaliana Col-0 genome (TAIR10) with STAR with the following settings: --runMode alignReads; --alignIntronMax 10000; --alignMatesGapMax 11000; --alignSJDBoverhangMin 1; --alignSJoverhangMin 10; --outSJfilterOverhangMin -1 10 10 10; --outSJfilterCountTotalMin -1 2 2 2; --outSJfilterCountUniqueMin -1 2 2 2; --outFilterType BySJout; --outFilterMismatchNmax 2; --outFilterMismatchNoverLmax .05; --twopassMode Basic; --outFilterIntronMotifs RemoveNoncanonicalUnannotated; --outFilterMultimapNmax 100; --outSAMmultNmax 100; --outSAMattributes NH HI AS nM NM MD jM jI XS; --outSAMprimaryFlag AllBestScore; --outSAMtype BAM Unsorted; --alignEndsType Local; --outFilterMatchNminOverLread 0.9. Aligned BAM files were used to calculate 5' end read depth for each position in the genome using the Python script readmapIO, which employs a "rich-get-richer" algorithm to assign reads that map to multiple locations, and outputs bedgraph files, which also include all nucleotides softclipped from the 5' ends of reads (upstream untemplated nucleotides (uuNs). Nucleotide-specific bias correction was applied as in Wang et al. (2017) BMC Bioinformatics with the following modifications. To apply the method to RNA-Seq data rather than DNA-Seq data, only the presence or absence of reads at a position was considered, not read abundance. K-mer frequencies of length 3 were calculated for reads mapping to the coding regions of TAIR10 genes, and bias correction against this k-mer matrix was applied to all reads.Discrete 5P features were identified from these alignments via subtractive kernel density estimation of the a sample's nanoPARE and mRNA-Seq libraries. Each feature was classified as capped if ≥10% of all reads from the sample contain uuG or classified as noncapped otherwise. Bedgraph files of 5P read positions relative to the start site of the dominant isoform of each gene in the reference annotation were prepared and capped features were masked for sRNA-mediated cleavage site analyses.
Genome_build: TAIR10
Supplementary_files_format_and_content: Bedgraph files contain tab-separated values of chromosomes or dominant transcripts (column 1), start (column 2) and stop (column 3) coordinates of 5' end of alignments and number of aligned ends weighted by their number of alignments.

source_name: unopened floral buds

extract_protocol:
TRIzol reagent (Thermo Fisher Scientific)
For Samples 1-6, mRNA-Seq libraries were generated according to the Smart-Seq2 protocol (Picelli et al. 2013. Nature Methods). For Samples 7-30, nanoPARE libraries were generated as detailed in the associated article.


title: 1ng_fb_3.nanoPARE

description: Single-end 50 base read nanoPARE data from third biological replicate of Col-0 flowers (1 ng)


title: col0_fb_10ug_3.nanoPARE

description: Single-end 50 base read nanoPARE data from third biological replicate of Col-0 flowers (10 µg)


title: flower_1.nanoPARE

description:
Single-end 50 base read nanoPARE data from first biological replicate of whole stage 13 Col-0 flowers
Sample 31


title: 100pg_fb_3.nanoPARE

description: Single-end 50 base read nanoPARE data from third biological replicate of Col-0 flowers (100 pg)


title: flower_2.nanoPARE

description:
Single-end 50 base read nanoPARE data from second biological replicate of whole stage 13 Col-0 flowers
Sample 32


title: 10pg_fb_2.nanoPARE

description: Single-end 50 base read nanoPARE data from second biological replicate of Col-0 flowers (10 pg)


title: 100pg_fb_2.nanoPARE

description: Single-end 50 base read nanoPARE data from second biological replicate of Col-0 flowers (100 pg)

data_processing:
Sequences in FASTQ files were trimmed with cutadapt, filtered for low-complexity and then aligned to the Arabopsis thaliana Col-0 genome (TAIR10) with STAR with the following settings: --runMode alignReads; --alignIntronMax 10000; --alignMatesGapMax 11000; --alignSJDBoverhangMin 1; --alignSJoverhangMin 10; --outSJfilterOverhangMin -1 10 10 10; --outSJfilterCountTotalMin -1 2 2 2; --outSJfilterCountUniqueMin -1 2 2 2; --outFilterType BySJout; --outFilterMismatchNmax 2; --outFilterMismatchNoverLmax .05; --twopassMode Basic; --outFilterIntronMotifs RemoveNoncanonicalUnannotated; --outFilterMultimapNmax 100; --outSAMmultNmax 100; --outSAMattributes NH HI AS nM NM MD jM jI XS; --outSAMprimaryFlag AllBestScore; --outSAMtype BAM Unsorted; --alignEndsType Local; --outFilterMatchNminOverLread 0.9. Aligned BAM files were used to calculate 5' end read depth for each position in the genome using the Python script readmapIO, which employs a "rich-get-richer" algorithm to assign reads that map to multiple locations, and outputs bedgraph files, which also include all nucleotides softclipped from the 5' ends of reads (upstream untemplated nucleotides (uuNs). Nucleotide-specific bias correction was applied as in Wange et al. (2017) BMC Bioinformatics with the following modifications. To apply the method to RNA-Seq data rather than DNA-Seq data, only the presence or absence of reads at a position was considered, not read abundance. K-mer frequencies of length 3 were calculated for reads mapping to the coding regions of TAIR10 genes, and bias correction against this k-mer matrix was applied to all reads.Discrete 5P features were identified from these alignments via subtractive kernel density estimation of the a sample's nanoPARE and mRNA-Seq libraries. Each feature was classified as capped if ≥10% of all reads from the sample contain uuG or classified as noncapped otherwise. Bedgraph files of 5P read positions relative to the start site of the dominant isoform of each gene in the reference annotation were prepared and capped features were masked for sRNA-mediated cleavage site analyses.
Genome_build: TAIR10
Supplementary_files_format_and_content: Bedgraph files contain tab-separated values of chromosomes or dominant transcripts (column 1), start (column 2) and stop (column 3) coordinates of 5' end of alignments and number of aligned ends weighted by their number of alignments.


title: 10pg_fb_1.nanoPARE

description: Single-end 50 base read nanoPARE data from first biological replicate of Col-0 flowers (10 pg)


title: 100pg_fb_1.nanoPARE

description: Single-end 50 base read nanoPARE data from first biological replicate of Col-0 flowers (100 pg)


title: 1ng_fb_2.nanoPARE

description: Single-end 50 base read nanoPARE data from second biological replicate of Col-0 flowers (1 ng)


title: 1ng_fb_1.nanoPARE

description: Single-end 50 base read nanoPARE data from first biological replicate of Col-0 flowers (1 ng)


title: col0_fb_10ug_2.nanoPARE

description: Single-end 50 base read nanoPARE data from second biological replicate of Col-0 flowers (10 µg)


title: dcl234_fb_3.nanoPARE

description: Single-end 50 base read nanoPARE data from third biological replicate of dcl234 triple mutant flowers

value: triple homozygous mutant with dcl2-1, dcl3-1, dcl4-2t alleles


title: col0_fb_10ug_1.nanoPARE

description: Single-end 50 base read nanoPARE data from first biological replicate of Col-0 flowers (10 µg)


title: xrn4_fb_3.nanoPARE

description: Single-end 50 base read nanoPARE data from third biological replicate of xrn4-5 flowers


title: dcl234_fb_1.nanoPARE

description: Single-end 50 base read nanoPARE data from first biological replicate of dcl234 triple mutant flowers


title: dcl234_fb_2.nanoPARE

description: Single-end 50 base read nanoPARE data from second biological replicate of dcl234 triple mutant flowers


title: col0_fb_1.nanoPARE

description: Single-end 50 base read nanoPARE data from first biological replicate of Col-0 flowers


title: xrn1_fb_3.nanoPARE

description: Single-end 50 base read nanoPARE data from third biological replicate of Col-0 flower RNA treated with Xrn1 prior to library construction


title: xrn4_fb_1.nanoPARE

description: Single-end 50 base read nanoPARE data from first biological replicate of xrn4-5 flowers


title: xrn1_fb_2.nanoPARE

description: Single-end 50 base read nanoPARE data from second biological replicate of Col-0 flower RNA treated with Xrn1 prior to library construction


title: xrn4_fb_2.mRNAseq

description: Single-end 50 base read poly(A) RNA-Seq  data from second biological replicate of xrn4-5 flowers

data_processing:
Sequences in FASTQ files were trimmed with cutadapt, filtered for low-complexity and then aligned to the Arabopsis thaliana Col-0 genome (TAIR10) with STAR with the following settings: --runMode alignReads; --alignIntronMax 10000; --alignMatesGapMax 11000; --alignSJDBoverhangMin 1; --alignSJoverhangMin 10; --outSJfilterOverhangMin -1 10 10 10; --outSJfilterCountTotalMin -1 2 2 2; --outSJfilterCountUniqueMin -1 2 2 2; --outFilterType BySJout; --outFilterMismatchNmax 2; --outFilterMismatchNoverLmax .05; --twopassMode Basic; --outFilterIntronMotifs RemoveNoncanonicalUnannotated; --outFilterMultimapNmax 100; --outSAMmultNmax 100; --outSAMattributes NH HI AS nM NM MD jM jI XS; --outSAMprimaryFlag AllBestScore; --outSAMtype BAM Unsorted; --alignEndsType EndToEnd. Aligned BAM files were used to calculate 5' end read depth for each position in the genome using the Python script readmapIO, which employs a "rich-get-richer" algorithm to assign reads that map to multiple locations, and outputs bedgraph files.
Genome_build: TAIR10
Supplementary_files_format_and_content: Bedgraph files contain tab-separated values of chromosomes or dominant transcripts (column 1), start (column 2) and stop (column 3) coordinates of 5' end of alignments and number of aligned ends weighted by their number of alignments.


title: xrn4_fb_1.mRNAseq

description: Single-end 50 base read poly(A) RNA-Seq data from first biological replicate of xrn4-5 flowers


title: xrn1_fb_1.nanoPARE

description: Single-end 50 base read nanoPARE data from first biological replicate of Col-0 flower RNA treated with Xrn1 prior to library construction


title: col0_fb_2.nanoPARE

description: Single-end 50 base read nanoPARE data from second biological replicate of Col-0 flowers


title: xrn4_fb_3.mRNAseq

description: Single-end 50 base read poly(A) RNA-Seq data from third biological replicate of xrn4-5 flowers


title: col0_fb_2.mRNAseq

description: Single-end 50 base read poly(A) RNA-Seq data from second biological replicate of Col-0 flowers

data_processing:
Sequences in FASTQ files were trimmed with cutadapt, filtered for low-complexity and then aligned to the Arabopsis thaliana Col-0 genome (TAIR10) with STAR with the following settings: --runMode alignReads; --alignIntronMax 10000; --alignMatesGapMax 11000; --alignSJDBoverhangMin 1; --alignSJoverhangMin 10; --outSJfilterOverhangMin -1 10 10 10; --outSJfilterCountTotalMin -1 2 2 2; --outSJfilterCountUniqueMin -1 2 2 2; --outFilterType BySJout; --outFilterMismatchNmax 2; --outFilterMismatchNoverLmax .05; --twopassMode Basic; --outFilterIntronMotifs RemoveNoncanonicalUnannotated; --outFilterMultimapNmax 100; --outSAMmultNmax 100; --outSAMattributes NH HI AS nM NM MD jM jI XS; --outSAMprimaryFlag AllBestScore; --outSAMtype BAM Unsorted; --alignEndsType EndToEnd. Aligned BAM files were used to calculate 5' end read depth for each position in the genome using the Python script readmapIO, which employs a "rich-get-richer" algorithm to assign reads that map to multiple locations, and outputs bedgraph files.
Supplementary_files_format_and_content: Bedgraph files contain tab-separated values of chromosomes or dominant transcripts (column 1), start (column 2) and stop (column 3) coordinates of 5' end of alignments and number of aligned ends weighted by their number of alignments.


title: col0_fb_1.mRNAseq

description: Single-end 50 base read poly(A) RNA-Seq data from first biological replicate of Col-0 flowers


title: col0_fb_3.mRNAseq

description: Single-end 50 base read poly(A) RNA-Seq data from third biological replicate of Col-0 flowers


title: 10APM_G4_n3

city: Toronto

first: Yu-Jia

last: Shiah

state: Ontario

address: MaRS Centre 661 University Avenue Suite 510

department: Informatics and Bio-computing

country: Canada

institute: Ontario Institute for Cancer Research

description: MYC10APM_G9a4vsSCR_DEResults_GeneAnnotated.txt

data_processing:
RNA-seq reads were mapped and quantified using STAR aligner (v2.5.2a) against GRCh37 with Gencode Annotations v24 lifed to GRCh37. Library normalization was performed using Trimmed Mean of M-values (TMM) from the BioConductor package edgeR (v3.14.0) in the R statistical environment (v3.3.1).
Differential expression analyses were conducted based on MYC target genes (n = 11,015) defined as being proximal to MYC binding regions as confirmed using non-specific knockdown (shSCR) MYC ChIP-seq data. Read counts of each specific knockdown were compared to read counts of shSCR using EBSeq (v1.16.0). Default parameters were used in the EBTest function except maxround was set to 10 iterations and normalized scores (quantile threshold = 0.7) from the read counts were used as sizeFactors. Given the Bayesian nature of EBSeq, one model was used to test all genes simultaneously so no additional multiple testing adjustment was made. Genes were deemed significantly differentially expressed if Posterior Probability of the genes being Differentially Expressed (PPDE) ≥ 0.95 which is equivalent to 1-PPDE < 0.05 and the measure is comparable to FDR < 0.05 in other test settings (e.g. DESeq method). log2PostFC = log2 posterior fold change which uses the posterior probabilities from a Bayesian model to calculate the fold change. For genes that have read counts = 0, no model was performed thus PPDE and PostFC = NA
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Read counts (.tab), differential expression analyses (.txt)

source_name: MCF10A cell line

growth_protocol: MCF10A cells were cultured in DMEM/F12 (1:1) media with 5% horse serum (HS) (Gibco), 0.1 μg/mL cholera toxin (Sigma), 0.02 μg/mL epidermal growth factor (EGF) (R&D Systems), 0.5 μg/mL hydrocortisone (Sigma), and 10 μg/mL insulin (Sigma).

extract_protocol:
RNA was extracted from subconfluent cells using the RNAeasy Mini Kit as per the manufacturer’s protocol (Qiagen). Sample quality was assessed using Agilent BioAnalyzer.
Sample libraries were prepared using TruSeq Stranded Total RNA Sample Preparation kit with Ribo Zero Gold (Illumina) and sequenced on Illumina NextSeq 500 with paired-end 75bp protocol multiplexed to achieve an average of 80M reads per sample at the Princess Margaret Genomics Centre (Toronto, Canada). PhiX spike-in at 0.5% per lane was included as control.

treatment_protocol: Asynchronously growing cells were seeded and treated with 2 μg/mL doxycycline (Sigma) for 72 hours to induce shRNA.

value: immortalized breast epithelial cells

tag: plasmid 1

value: pBABE-hygro-MYC

tag: plasmid 2

tag: plasmid 3

value: pLKO-tetON-puro-shG9a 4


title: 10APM_G4_n2


title: 10APM_G1_n2

description: MYC10APM_G9a1vsSCR_DEResults_GeneAnnotated.txt

value: pLKO-tetON-puro-shG9a 1


title: 10APM_G1_n3


title: 10APM_G4_n1


title: col0_fb_3.nanoPARE

description: Single-end 50 base read nanoPARE data from third biological replicate of Col-0 flowers


title: 10APM_S_n2

value: pLKO-tetON-puro-shSCR


title: xrn4_fb_2.nanoPARE

description: Single-end 50 base read nanoPARE data from second biological replicate of xrn4-5 flowers


title: 10APM_G1_n1


title: 10APM_S_n3


title: 1741_014_v1_HFWB_TPM_X27hr

city: Chicago

first: Marta

last: Iwanaszko

state: IL

address: 303 E. Superior Street, Simpson Querrey 7th Floor,

department: Biochemistry and Molecular Genetics

country: USA

institute: Northwestern University Feinberg School of Medicine

data_processing:
Transcripts were aligned and quantified using STAR (version 2.5.2) and RSEM (version 1.2.31).
STAR was used with the options - -quantMode TranscriptomeSAM GeneCounts –outSAMtype BAM SortedByCoordinate to produce bam files with reads aligned to both the transcriptome and the reference.
RSEM was used with options - -bam –paired-end –forward-prob 0 –no-bam-output to produce tags-per-million (TPM) counts for each gene from transcriptome alignments.
Quality control assessment was performed computing transcript integrity numbers for each gene, and average transcript integrity numbers for each sample, using tin.py from RSeQC. Postprocessing of the count and quality data into table form was performed with custom Perl, Python and shell scripts.
Genome_build: Gencode22 (GRCh38.primary_assembly.genome.fa, gencode.v22.primary_assembly.annotation.gtf)
Supplementary_files_format_and_content: Comma-delimited text file including TPMs for all samples

source_name: Whole blood

extract_protocol:
At each timepoint for each subject, 2.5 mL of whole blood was drawn into a PAXgene RNA tube and processed according to manufacturer protocol (BD Vacutainer). RNA was extracted using the PAXgene Blood RNA Kit (Qiagen), with an expected yield of 3 µ g total RNA from 2.5 mL blood. RNA was then subject to globin transcript reduction and stored at -80C until processing for Illumina RNA-seq.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Whole blood

tag: subjectid

tag: hour


title: 1741_014_v1_HFWB_TPM_X23hr


title: 1741_014_v1_HFWB_TPM_X25hr


title: 1741_014_v1_HFWB_TPM_X19hr


title: 1741_014_v1_HFWB_TPM_X15hr


title: 1741_014_v1_HFWB_TPM_X09hr


title: 1741_014_v1_HFWB_TPM_X05hr


title: 1741_014_v1_HFWB_TPM_X01hr


title: 1741_011_v1_HFWB_TPM_X27hr


title: 1741_011_v1_HFWB_TPM_X25hr


title: 1741_014_v1_HFWB_TPM_X07hr


title: 1741_011_v1_HFWB_TPM_X21hr


title: 1741_014_v1_HFWB_TPM_X03hr


title: 1741_011_v1_HFWB_TPM_X23hr


title: 1741_011_v1_HFWB_TPM_X17hr


title: 10APM_S_n1


title: 1741_011_v1_HFWB_TPM_X15hr


title: 1741_011_v1_HFWB_TPM_X09hr


title: 1741_011_v1_HFWB_TPM_X13hr


title: 1741_011_v1_HFWB_TPM_X11hr


title: 1741_014_v1_HFWB_TPM_X21hr


title: 1741_011_v1_HFWB_TPM_X07hr


title: 1741_011_v1_HFWB_TPM_X01hr


title: 1741_014_v1_HFWB_TPM_X11hr


title: 1741_011_v1_HFWB_TPM_X05hr


title: 1741_011_v1_HFWB_TPM_X03hr


title: 1741_010_v1_HFWB_TPM_X25hr


title: 1741_011_v1_HFWB_TPM_X19hr


title: 1741_010_v1_HFWB_TPM_X23hr


title: 1741_010_v1_HFWB_TPM_X13hr


title: 1741_014_v1_HFWB_TPM_X13hr


title: 1741_010_v1_HFWB_TPM_X17hr


title: 1741_010_v1_HFWB_TPM_X19hr


title: 1741_010_v1_HFWB_TPM_X21hr


title: 1741_010_v1_HFWB_TPM_X11hr


title: 1741_010_v1_HFWB_TPM_X09hr


title: 1741_010_v1_HFWB_TPM_X05hr


title: 1741_010_v1_HFWB_TPM_X03hr


title: 1741_010_v1_HFWB_TPM_X01hr


title: 1741_010_v1_HFWB_TPM_X07hr


title: 1741_009_v1_HFWB_TPM_X25hr


title: 1741_009_v1_HFWB_TPM_X23hr


title: 1741_010_v1_HFWB_TPM_X15hr


title: 1741_010_v1_HFWB_TPM_X27hr


title: 1741_009_v1_HFWB_TPM_X15hr


title: 1741_009_v1_HFWB_TPM_X19hr


title: 1741_009_v1_HFWB_TPM_X13hr


title: 1741_009_v1_HFWB_TPM_X11hr


title: 1741_009_v1_HFWB_TPM_X21hr


title: 1741_009_v1_HFWB_TPM_X05hr


title: 1741_009_v1_HFWB_TPM_X17hr


title: 1741_009_v1_HFWB_TPM_X09hr


title: 1741_009_v1_HFWB_TPM_X03hr


title: 1741_006_v1_HFWB_TPM_X17hr


title: 1741_006_v1_HFWB_TPM_X25hr


title: 1741_006_v1_HFWB_TPM_X19hr


title: 1741_006_v1_HFWB_TPM_X23hr


title: 1741_006_v1_HFWB_TPM_X11hr


title: 1741_009_v1_HFWB_TPM_X27hr


title: 1741_006_v1_HFWB_TPM_X15hr


title: 1741_006_v1_HFWB_TPM_X21hr


title: 1741_006_v1_HFWB_TPM_X13hr


title: 1741_009_v1_HFWB_TPM_X07hr


title: 1741_006_v1_HFWB_TPM_X07hr


title: 1741_006_v1_HFWB_TPM_X05hr


title: 1741_006_v1_HFWB_TPM_X03hr


title: 1741_006_v1_HFWB_TPM_X09hr


title: 1741_005_v1_HFWB_TPM_X23hr


title: 1741_005_v1_HFWB_TPM_X25hr


title: 1741_009_v1_HFWB_TPM_X01hr


title: 1741_005_v1_HFWB_TPM_X21hr


title: 1741_005_v1_HFWB_TPM_X19hr


title: ZNF787OE.d4.3 (ZNF787)

city: Dresden

first: Volker

last: Busskamp

state: Saxony

address: Fetscherstraße 105

department: CMCB

institute: Technische Universität Dresden

data_processing:
Whole-cell RNA-Seq data reads were mapped to the human reference genome (hg38) using GSNAP (Wu and Nacu, 2010) with ENSEMBL annotation 81.
Quality control with FASTQC (bioinformatics.babraham.ac.uk/projects/fastqc/, v0.11.4, accessed 2016-09-10)
Reads were counted using featureCounts (Liao et al., 2014, version 1.5.2).
Differential expression analysis was performed using DESeq2 (Love et al., 2014).
Genome_build: hg38
Supplementary_files_format_and_content: RNA-Seq_ZNF787_Supplement.xlsx comprises raw counts per gene for each sample as a spreadsheet.

source_name: iNGN cells (hiPSCs with Neurog1/2 cassette)

growth_protocol: hiPSCs were cultured in mTeSR 1 media (05850, StemCell Technologies) on standard tissue culture plates coated with hESC-qualified Matrigel matrix (354277, Corning, incubated 45 min at room temperature). For passaging, hiPSCs were dissociated with TrypLE Express (12604013, Thermo Fisher Scientific), washed with DPBS (pH 7.2; 14190169, Thermo Fisher Scientific), spun down (400 × g) and replated using mTESR with 3 µg/ml InSolution Y-27632 rho kinase inhibitor (688001, Merck Millipore). 0.5 µg/ml doxycycline (D9891, Sigma-Aldrich) was used to induce Neurog1 and -2 genes for four days (referred to as days post induction/dpi).

extract_protocol:
At 4 dpi, cells were detached with TrypLE Express (12604013, Thermo Fisher Scientific), spun down, and lysed with Qiazol (Qiagen). The phases were separated with Phase Lock Gel tubes (heavy, 2 ml, 733-2478, 5PRIME) and the aqueous phase was cleaned-up with the miRNeasy Kit (Qiagen).
cDNA libraries were constructed using NEBNext Ultra I Directional RNA Library Prep Kit for Illumina (E7420, NEB) with 1 µg input. PolyA selection, fragmentation, first strand and second strand cDNA synthesis, and purification were performed in combination with Agencourt AMPure Kit (A63880, Beckman Coulter) and repair/dA-tailing of the cDNA. Adapters were ligated to dA-tailed cDNA and size-selected using AMPure XP Beads (Beckman Coulter). Index primers (Illumina) were used to index library constructs, and a PCR was performed using NEBNext Q5 2X PCR Master Mix (M0543S, NEB). Libraries were purified using Agencourt AMPure Kit. Libraries were pooled and sequenced on a HiSeq 2500 (Illumina).

treatment_protocol: For the ∆miR-124 line, sgRNA were designed by the MIT’s CRISPR web tool (http://crispr.mit.edu/; Hsu et al., 2013). sgRNA sequences and corresponding oligos. All oligos were cloned into the vector pSpCas9(BB)-2A-Puro (PX459) V2.0 (a kind gift from Feng Zhang, 62988, Addgene) as described in Ran et al., 2013. Four different sgRNA-expressing constructs were co-electroporated (10 µg per vector) into iNGN and CRTD5 cells by the Lonza 4D X-unit, pulse CB-156, and the P3 Primary Cell 4D-Nucleofector Kit L (V4XP-3024, Lonza), according to the manufacturer’s guidelines. Cells were plated sparsely, and selected for PX459 integration after 24h with puromycin. Grown colonies, derived from single cells, were picked and genotyped with primers specific for all miR-124 loci. All six genomic miR-124 copies were depleted. ZNF787 overexpression was performed using the PiggyBac vector PB-TRE-dCas9-VPR13 (Addgene plasmid #63800; Chavez et al., 2015) with addition of a doxycycline-inducible promoter, PCR-amplified ZNF787 (variant 1), a V5 tag and a blasticidin resistance gene after via isothermal assembly (construct PB09-TRE-ZNF787-V5-Blasti). Stable integration was assured by administration of 15 µg/ml blasticidin for at least three days.

value: ZNF787 OE

tag: passage

tag: day post induction


title: miR124KO.d4.1 (ZNF787)


title: WT.d4.3 (ZNF787)

value: WT


title: ZNF787OE.d4.2 (ZNF787)


title: miR124KO.d4.2 (ZNF787)


title: WT.d4.2 (ZNF787)


title: 1741_005_v1_HFWB_TPM_X11hr


title: WT.d4.1 (ZNF787)


title: 1741_006_v1_HFWB_TPM_X01hr


title: 1741_005_v1_HFWB_TPM_X15hr


title: 1741_005_v1_HFWB_TPM_X05hr


title: 1741_005_v1_HFWB_TPM_X27hr


title: 1741_005_v1_HFWB_TPM_X13hr


title: ZNF787OE.d4.1 (ZNF787)


title: miR124KO.d4.3 (ZNF787)


title: miR124KO_d14_2 (d7+d14)

data_processing:
Reads were adapter-clipped using samtools, version 1.3 (http://www.htslib.org/).
Quality control was performed with FASTQC (bioinformatics.babraham.ac.uk/projects/fastqc/, v0.11.4)
Reads were mapped to the human genome assembly hg38/GRCh38 using Segemehl (v0.2.0-418, Hoffmann et al., 2009).
Counts were computed using rnacounter (https://github.com/jdelafon/rnacounter) using RPKM and raw counts, from the v25 gencode annotation.
Genome_build: hg38
Supplementary_files_format_and_content: RNA-Seq_7+14dpi_Supplement.xlsx comprises raw counts per gene for each sample as a spreadsheet.

growth_protocol: hiPSCs were cultured in mTeSR 1 media (05850, StemCell Technologies) on standard tissue culture plates coated with hESC-qualified Matrigel matrix (354277, Corning, incubated 45 min at room temperature). For passaging, hiPSCs were dissociated with TrypLE Express (12604013, Thermo Fisher Scientific), washed with DPBS (pH 7.2; 14190169, Thermo Fisher Scientific), spun down (400 × g) and replated using mTESR with 3 µg/ml InSolution Y-27632 rho kinase inhibitor (688001, Merck Millipore). 0.5 µg/ml doxycycline (D9891, Sigma-Aldrich) was used to induce Neurog1 and -2 genes for a maximum of four days (referred to as days post induction/dpi).

extract_protocol:
at 7 or 14 dpi, cells were detached with TrypLE Express (12604013, Thermo Fisher Scientific), spun down, and lysed with Qiazol (Qiagen). The phases were separated with Phase Lock Gel tubes (heavy, 2 ml, 733-2478, 5PRIME) and the aqueous phase was cleaned-up with the miRNeasy Kit (Qiagen).
cDNA libraries were prepared using NEBNext Ultra I Directional RNA Library Prep Kit for Illumina (E7420, NEB) with 500 ng input. PolyA selection, fragmentation, first strand and second strand cDNA synthesis, and purification were performed in combination with Agencourt AMPure Kit (A63880, Beckman Coulter) and repair/dA-tailing of the cDNA. Adapters were ligated to dA-tailed cDNA and size-selected using AMPure XP Beads (Beckman Coulter). Index primers (Illumina) were used to index library constructs, and a PCR was performed using NEBNext Q5 2X PCR Master Mix (M0543S, NEB). Libraries were purified using Agencourt AMPure Kit.

treatment_protocol: 250,000 cells of each sample group, WT and ∆miR-124 (KO) were seeded onto Matrigel-coated 12-well plates and induced with doxycycline for a maximum of four days. After 4 dpi, half of the mTeSR 1 media was replaced with supplemented BrainPhys media (05790, StemCell Technologies), replaced daily until 7 dpi and further once per week. The following BrainPhys supplements were used for 10 ml media: 200 µl NeuroCult™ SM1 Neuronal Supplement (05711, Stemcell Technologies); 100 µl N2 Supplement-A (17502048, Thermo Fisher Scientific); 20 µl of 10 µg/ml recombinant human BDNF to a final concentration of 20 ng/ml (450-02, Peprotech); 20 µl of 10 µg/ml recombinant human GDNF to a final concentration of 20 ng/ml (450-10, Peprotech); 98 µl of 50 mg/ml dibutryl-cAMP to a final concentration of 1 mM (D0627, Sigma-Aldrich); 50 µl of 40 mM ascorbic acid to a final concentration of 200 nM (A0278, Sigma-Aldrich); and 100 µl of 100× penicillin-streptomycin (15140122, Thermo Fisher Scientific).


title: miR124KO_d14_3 (d7+d14)


title: miR124KO_d14_1 (d7+d14)


title: WT_d14_3 (d7+d14)


title: 1741_005_v1_HFWB_TPM_X03hr


title: 1741_005_v1_HFWB_TPM_X01hr


title: WT_d14_2 (d7+d14)


title: 1741_001_v1_HFWB_TPM_X25hr


title: WT_d14_1 (d7+d14)


title: miR124KO_d7_3 (d7+d14)


title: 1741_005_v1_HFWB_TPM_X17hr


title: miR124KO_d7_2 (d7+d14)


title: 1741_005_v1_HFWB_TPM_X07hr


title: 1741_001_v1_HFWB_TPM_X23hr


title: miR124KO_d7_1 (d7+d14)


title: WT_d7_3 (d7+d14)


title: 1741_001_v1_HFWB_TPM_X21hr


title: WT_d7_2 (d7+d14)


title: WT_d7_1 (d7+d14)


title: 1741_001_v1_HFWB_TPM_X19hr


title: 1741_001_v1_HFWB_TPM_X17hr


title: 1741_001_v1_HFWB_TPM_X13hr


title: 1741_001_v1_HFWB_TPM_X11hr


title: 1741_001_v1_HFWB_TPM_X27hr


title: KO.d4.TC.7 (TimeCourse)

data_processing:
Reads were adapter-clipped using samtools, version 1.3 (http://www.htslib.org/).
Quality control was performed with FASTQC (bioinformatics.babraham.ac.uk/projects/fastqc/, v0.11.4)
Reads were mapped to the human genome assembly hg38/GRCh38 using Segemehl (v0.2.0-418, Hoffmann et al., 2009).
Counts were computed using rnacounter (https://github.com/jdelafon/rnacounter) using RPKM and raw counts, from the v25 gencode annotation.
Genome_build: hg38
Supplementary_files_format_and_content: RNA-Seq_timecourse_Supplement.xlsx includes raw counts per gene for each sample as a spreadsheet.

extract_protocol:
At 4 dpi, cells were detached with TrypLE Express (12604013, Thermo Fisher Scientific), spun down, and lysed with Qiazol (Qiagen). The phases were separated with Phase Lock Gel tubes (heavy, 2 ml, 733-2478, 5PRIME) and the aqueous phase was cleaned-up with the miRNeasy Kit (Qiagen).
cDNA libraries were prepared using NEBNext Ultra I Directional RNA Library Prep Kit for Illumina (E7420, NEB) with 500 ng input. PolyA selection, fragmentation, first strand and second strand cDNA synthesis, and purification were performed in combination with Agencourt AMPure Kit (A63880, Beckman Coulter) and repair/dA-tailing of the cDNA. Adapters were ligated to dA-tailed cDNA and size-selected using AMPure XP Beads (Beckman Coulter). Index primers (Illumina) were used to index library constructs, and a PCR was performed using NEBNext Q5 2X PCR Master Mix (M0543S, NEB). Libraries were purified using Agencourt AMPure Kit.

treatment_protocol: 250,000 cells of each sample group, WT and ∆miR-124 (KO) were seeded onto Matrigel-coated 12-well plates and induced with doxycycline for a maximum of four days.


title: KO.d4.TC.6 (TimeCourse)


title: KO.d4.TC.5 (TimeCourse)


title: KO.d4.TC.4 (TimeCourse)


title: 1741_001_v1_HFWB_TPM_X09hr


title: 1741_001_v1_HFWB_TPM_X07hr


title: KO.d4.TC.2 (TimeCourse)


title: KO.d4.TC.3 (TimeCourse)


title: KO.d4.TC.1 (TimeCourse)


title: KO.d3.TC.8 (TimeCourse)


title: 1674_007_v1_HFWB_TPM_X29hr


title: KO.d3.TC.7 (TimeCourse)


title: 1674_007_v1_HFWB_TPM_X27hr


title: 1741_001_v1_HFWB_TPM_X15hr


title: KO.d3.TC.5 (TimeCourse)


title: 1741_001_v1_HFWB_TPM_X01hr


title: KO.d3.TC.6 (TimeCourse)


title: 1674_007_v1_HFWB_TPM_X25hr


title: 1674_007_v1_HFWB_TPM_X23hr


title: KO.d3.TC.2 (TimeCourse)


title: KO.d3.TC.4 (TimeCourse)


title: KO.d3.TC.1 (TimeCourse)


title: 1674_007_v1_HFWB_TPM_X21hr


title: 1741_001_v1_HFWB_TPM_X03hr


title: 1741_001_v1_HFWB_TPM_X05hr


title: 1674_007_v1_HFWB_TPM_X15hr


title: KO.d2.TC.8 (TimeCourse)


title: KO.d2.TC.6 (TimeCourse)


title: KO.d2.TC.5 (TimeCourse)


title: KO.d2.TC.7 (TimeCourse)


title: 1674_007_v1_HFWB_TPM_X07hr


title: 1674_007_v1_HFWB_TPM_X09hr


title: 1674_007_v1_HFWB_TPM_X13hr


title: 1674_007_v1_HFWB_TPM_X11hr


title: KO.d2.TC.4 (TimeCourse)


title: 1674_007_v1_HFWB_TPM_X03hr


title: 1674_007_v1_HFWB_TPM_X05hr


title: KO.d2.TC.3 (TimeCourse)


title: KO.d2.TC.2 (TimeCourse)


title: 1674_007_v1_HFWB_TPM_X01hr


title: KO.d1.TC.8 (TimeCourse)


title: 1674_006_v1_HFWB_TPM_X29hr


title: KO.d1.TC.7 (TimeCourse)


title: 1674_006_v1_HFWB_TPM_X25hr


title: KO.d1.TC.6 (TimeCourse)


title: 1674_007_v1_HFWB_TPM_X17hr


title: KO.d1.TC.5 (TimeCourse)


title: 1674_006_v1_HFWB_TPM_X23hr


title: 1674_006_v1_HFWB_TPM_X17hr


title: KO.d1.TC.3 (TimeCourse)


title: KO.d1.TC.4 (TimeCourse)


title: KO.d0.TC.8 (TimeCourse)


title: 1674_006_v1_HFWB_TPM_X19hr


title: KO.d1.TC.2 (TimeCourse)


title: 1674_006_v1_HFWB_TPM_X15hr


title: KO.d0.TC.7 (TimeCourse)


title: 1674_006_v1_HFWB_TPM_X21hr


title: 1674_006_v1_HFWB_TPM_X09hr


title: 1674_006_v1_HFWB_TPM_X11hr


title: KO.d0.TC.6 (TimeCourse)


title: 1674_006_v1_HFWB_TPM_X13hr


title: 1674_006_v1_HFWB_TPM_X05hr


title: 1674_006_v1_HFWB_TPM_X07hr


title: KO.d0.TC.4 (TimeCourse)


title: KO.d0.TC.3 (TimeCourse)


title: 1674_005_v1_HFWB_TPM_X29hr


title: KO.d0.TC.2 (TimeCourse)


title: 1674_006_v1_HFWB_TPM_X01hr


title: 1674_006_v1_HFWB_TPM_X03hr


title: WT.d4.TC.8 (TimeCourse)


title: WT.d4.TC.4 (TimeCourse)


title: WT.d4.TC.6 (TimeCourse)


title: WT.d4.TC.7 (TimeCourse)


title: 1674_005_v1_HFWB_TPM_X21hr


title: 1674_005_v1_HFWB_TPM_X25hr


title: 1674_005_v1_HFWB_TPM_X27hr


title: WT.d4.TC.3 (TimeCourse)


title: 1674_005_v1_HFWB_TPM_X13hr


title: 1674_005_v1_HFWB_TPM_X11hr


title: 1674_005_v1_HFWB_TPM_X23hr


title: 1674_005_v1_HFWB_TPM_X19hr


title: WT.d3.TC.6 (TimeCourse)


title: WT.d3.TC.7 (TimeCourse)


title: WT.d4.TC.2 (TimeCourse)


title: WT.d3.TC.4 (TimeCourse)


title: 1674_005_v1_HFWB_TPM_X09hr


title: WT.d4.TC.1 (TimeCourse)


title: WT.d3.TC.8 (TimeCourse)


title: WT.d2.TC.8 (TimeCourse)


title: WT.d3.TC.5 (TimeCourse)


title: 1674_005_v1_HFWB_TPM_X03hr


title: 1674_005_v1_HFWB_TPM_X05hr


title: 1674_005_v1_HFWB_TPM_X15hr


title: WT.d3.TC.2 (TimeCourse)


title: 1674_001_v1_HFWB_TPM_X27hr


title: WT.d2.TC.5 (TimeCourse)


title: WT.d2.TC.3 (TimeCourse)


title: WT.d2.TC.2 (TimeCourse)


title: 1674_001_v1_HFWB_TPM_X23hr


title: 1674_005_v1_HFWB_TPM_X07hr


title: WT.d2.TC.6 (TimeCourse)


title: WT.d1.TC.8 (TimeCourse)


title: 1674_005_v1_HFWB_TPM_X01hr


title: 1674_001_v1_HFWB_TPM_X21hr


title: WT.d2.TC.7 (TimeCourse)


title: 1674_001_v1_HFWB_TPM_X19hr


title: 1674_001_v1_HFWB_TPM_X15hr


title: 1674_005_v1_HFWB_TPM_X17hr


title: 1674_001_v1_HFWB_TPM_X29hr


title: 1674_001_v1_HFWB_TPM_X11hr


title: WT.d3.TC.1 (TimeCourse)


title: 1674_001_v1_HFWB_TPM_X17hr


title: WT.d1.TC.1 (TimeCourse)


title: 1674_001_v1_HFWB_TPM_X05hr


title: 1674_001_v1_HFWB_TPM_X13hr


title: WT.d0.TC.7 (TimeCourse)


title: WT.d2.TC.1 (TimeCourse)


title: WT.d1.TC.5 (TimeCourse)


title: WT.d0.TC.6 (TimeCourse)


title: 1674_001_v1_HFWB_TPM_X03hr


title: WT.d1.TC.2 (TimeCourse)


title: WT.d1.TC.3 (TimeCourse)


title: WT.d1.TC.6 (TimeCourse)


title: 1674_001_v1_HFWB_TPM_X25hr


title: 1674_001_v1_HFWB_TPM_X07hr


title: WT.d0.TC.3 (TimeCourse)


title: WT.d0.TC.8 (TimeCourse)


title: 72--LLC 2wks cancer progression

city: Fayetteville

first: Nicholas

middle: P.

last: Greene

state: Arkansas

address: 308 HPER Building

department: Health, Human Performance and Recreation

institute: University of Arkansas

description: LLC cancer WK2

data_processing:
Sequenced reads were performed quality assessment using FastQC.
Sequenced reads were then aligend to GRCm38.p5 reference genome using Hisat2 tool.
Raw read counts were then obtained from aligned files using HTSeq.
Differential expression was analyzed by DESeq2
Genome_build: GRCm38.p5
Supplementary_files_format_and_content: Test files include raw read counts for each sample and expression data.

organism: Mus musculus

source_name: gastrocnemius muscle

extract_protocol:
Gastrocnemius muscles were removed, flash frozen in liquid nitrogen, and RNA was harvested using Trizol reagent.
Illumina TruSeq RNA Library Prep Kit was used with 1 ug of total RNA for the construction of sequencing libraries.

tag: strain

value: gastrocnemius muscle

tag: tumor development

value: 14 days


title: 1674_001_v1_HFWB_TPM_X01hr


title: WT.d0.TC.4 (TimeCourse)


title: 56--LLC 3wks cancer progression

description: LLC cancer WK3

value: 21 days


title: Hi-C_HepG2

city: Saarbrücken

first: Abdulrahman

last: Salhab

address: campus Saarbrücken

department: Genetics

institute: Saarland university

data_processing:
Reads were mapped to the human reference genome hg19 (37d5) using bowtie2, and then samtools was used to convert the reads to BAM format. A matrix of read counts over the bins in the genome, considering the sites around the restriction site AAGCTT was built using the hicBuildMatrix function from HiCExplorer. Ten bins were merged with hicMergeMatrixBins and then the matrix was corrected for GC bias and very low/high contact regions. To compute the TADs we first calculated the TAD scores by “hicFindTADs TAD_score” command with the following parameters “–minDepth 300000 –maxDepth 2000000 –step 70000” and then TADs were identified by “hicFindTADs find_TADs” command. The interaction matrix and other signal tracks were also visualized using HiCExplorer.
Genome_build: hg19
Supplementary_files_format_and_content: matrix containing the Hi-C contacts

source_name: HepG2 cell line

extract_protocol:
Hi-C experiments have been conducted as previously described (Ramírez, Fidel, et al. "High-resolution TADs reveal DNA sequences underlying genome organization in flies." Nature communications 9.1 (2018): 189.), with the following modifications. Nuclei from cell pellets containing about 4 million of cells have been extracted by sonication (Arrigoni, Laura, et al. "Standardizing chromatin research: a simple and universal method for ChIP-seq." Nucleic acids research 44.7 (2015): e67-e67.) using the following parameters: 75W peak power, 2% duty factor, 200 cycles/burst, 180 seconds, using Covaris milliTubes and Covaris E220 sonicator. After nuclei permeabilization, chromatin has been digested overnight at $37ºC using HindIII high fidelity (80 units per million cells; R3104S, NEB). Biotin incorporation has been carried out at 37ºC for one hour in 300 μl volume using these reaction conditions: 50 mM of each nucleotide (dATP, dTTP, dGTP, biotin-14-dCTP, from Life Technologies, 19518-018), 8 U of Klenow (NEB, M0210L). Ligase mix has been added to each sample followed by 4 hours of incubation at room temperature under rotation. After nuclei lysis, protein digestion and overnight de-crosslink, DNA has been precipitated and sonicated to 100-600 bp. Biotinylated DNA has been pulled down as previously described.
100 ng of DNA bound to beads have been used for library preparation using a modification of the NEBNext Ultra DNA library preparation workflow (NEB, E7370). DNA bound to beads has been end-repaired, A-tailed, adaptor-ligated and USER-treated following manufacturer’s instruction. After a bead wash, DNA has been eluted from the beads by incubating at 98°C for 10 minutes. Adaptor-ligated DNA has been PCR amplified using 7 PCR cycles. Libraries have been sequenced paired-end, with a read length of 75 bp, on the Illumina NextSeq 500 instrument.

tag: cell type

value: Hepatocellular carcinoma


title: 1674_001_v1_HFWB_TPM_X09hr


title: 41-PBS CONTROL

description: LLC cancer WK0

value: 0 days


title: 73--LLC 2wks cancer progression

description: LLC cancer WK2


title: WT.d1.TC.7 (TimeCourse)


title: 59--LLC 3wks cancer progression

description: LLC cancer WK3


title: 37-PBS CONTROL

description: LLC cancer WK0


title: WT.d0.TC.2 (TimeCourse)


title: WT.d0.IN.3 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample

data_processing:
RIP-Seq reads were pre-processed (includes demultiplexing, barcode trimming, and adaptor removal) using BBDuk from the BBMap toolkit (github.com/BioInfoTools/BBMap, v36.14, accessed 2016-08-15).
Quality control with FASTQC (bioinformatics.babraham.ac.uk/projects/fastqc/, v0.11.4, accessed 2016-09-10)
Reads were mapped to the human genome assembly hg38/GRCh38 using Segemehl (v0.2.0-418, Hoffmann et al., 2009). Uniquely mapped reads were extracted for further analysis.
For differential expression analysis of the whole-cell RNA-Seq data reads were mapped to the human reference genome (hg38) using GSNAP (Wu and Nacu, 2010; accessed 2017-03-17) with ENSEMBL annotation 81 (July 2015) and counted using featureCounts (Liao et al., 2014, version 1.5.2). Differential expression analysis was performed using DESeq2 (Love et al., 2014) on uniquely mapped reads.
The AGO2-IP signal was normalized with the gene expression, which was used to compare relative levels of mRNAs and miRNAs(separate upload) associated with the RISC complex between WT and ΔmiR-124 cells. We used a custom filtering method to split potential binding regions once certain height thresholds were reached. Cutoffs were defined based on signals detected in known miR-124 targets. Regions with a summit signal below 10 read pile-ups are considered background and were discarded. With a sliding window approach, starting from the summit, a binding site is first split when its height falls below 20 % of the summit signal. Emerging sub-sites with a summit above this cutoff and 10 pile-ups are then recursively split when their signal falls below 5 % of their summit, which also preserves regions with only weak signal. Replicates of each experimental setup were analyzed separately, and only peaks occurring in all replicates were considered for further analysis. Binding sites derived from uniquely mapped reads were annotated using the ENSEMBL Perl API for human annotation v90.
Genome_build: hg38
Supplementary_files_format_and_content: RIP_Supplement: Excel spreadsheet with (Table S7) raw counts from mapping with GSNAP, (Table S8) peak signal detected for the 3' UTR for AGO2-immunoprecipation samples (as 3' UTR score/gene)

extract_protocol:
Cell pellets from approximately 50 million cells were homogenized, lysed in ice-cold lysis buffer (10 mM HEPES [pH 7.3, ThermoFisher Scientific], 100 mM KCl [1049360250, Merck], 0.5 % NP40 [NP40S, Sigma-Aldrich], 5 mM MgCl2, 0.5 mM dithiothreitol [DTT, DTT-RO, Sigma-Aldrich], protease inhibitors [118735, Sigma-Aldrich], recombinant RNase inhibitors [10777019, ThermoFisher Schientific], 1 mM PMSF [36978, ThermoFisher Scientific]) using TissueLyser LT (50 Hz, 2 min, Qiagen). For lysate clearing, homogenates were centrifuged for 15 min at 16,200 × g, 4°C. A 50 µl sample was taken as a pre-IP sample. The RIP samples were incubated with Dynabeads Protein G beads (10009D, ThermoFisher Scientific) coated with anti-hAGO2 antibody (a gift from Jay Nelson; Grey et al., 2010) for 24h at 4°C with end-over-end rotation. After incubation, beads were collected on a Dynamagnet (1 min, on ice, ThermoFisher Scientific) and gently resuspended in low-salt NT2 buffer (50 mM Tris-HCl [pH 7.5, T1503, Sigma-Aldrich], 1 mM MgCl2, 150 mM NaCl [S3014, Sigmal-Aldrich], 0.5 % NP40, 0.5 mM dithiothreitol, 1 mM PMSF, protease inhibitors, recombinant RNase inhibitors). The beads were transferred to a new collection tube and washed with low salt NT2 buffer, followed by two washes with high salt NT2 buffer (50 mM Tris-HCl [pH 7.5], 1 mM MgCl2, 600 mM NaCl, 0.5 % NP40, 0.5 mM DTT, protease inhibitors, 1 mM PMSF, recombinant RNase inhibitors). The solution containing the RNA was then transferred to Qiazol (Qiagen). Subsequently, RNA was isolated from RIP and pre-RIP samples according to the miRNeasy micro kit protocol (Qiagen). The size distribution of the samples and the quality was checked with a Bioanalyzer RNA 6000 Pico Chip (Agilent Genomics).
cDNA libraries of three independent WT whole-cell RNA, ∆miR-124 whole-cell RNA, WT-AGO2 RIP, and ΔmiR-124-AGO2 RIP samples for 0 and 4 dpi were prepared using NEBNext Ultra I Directional RNA Library Prep Kit for Illumina (E7420, NEB) with 900 ng input. PolyA selection, fragmentation, first strand and second strand cDNA synthesis, and purification were performed in combination with Agencourt AMPure Kit (A63880, Beckman Coulter) and repair/dA-tailing of the cDNA. Adapters were ligated to dA-tailed cDNA and size-selected using AMPure XP Beads (Beckman Coulter). RIP samples were not polyA-selected. Index primers (Illumina) were used to index library constructs, and a PCR was performed using NEBNext Q5 2X PCR Master Mix (M0543S, NEB). Libraries were purified using Agencourt AMPure Kit. Libraries were pooled and sequenced on a HiSeq 2500 (Illumina), resulting in ca. 33-56 million single-end 75 bp reads.

treatment_protocol: For the ∆miR-124 line, sgRNA were designed by the MIT’s CRISPR web tool (http://crispr.mit.edu/; Hsu et al., 2013). sgRNA sequences and corresponding oligos. All oligos were cloned into the vector pSpCas9(BB)-2A-Puro (PX459) V2.0 (a kind gift from Feng Zhang, 62988, Addgene) as described in Ran et al., 2013. Four different sgRNA-expressing constructs were co-electroporated (10 µg per vector) into iNGN and CRTD5 cells by the Lonza 4D X-unit, pulse CB-156, and the P3 Primary Cell 4D-Nucleofector Kit L (V4XP-3024, Lonza), according to the manufacturer’s guidelines. Cells were plated sparsely, and selected for PX459 integration after 24h with puromycin. Grown colonies, derived from single cells, were picked and genotyped with primers specific for all miR-124 loci. All six genomic miR-124 copies were depleted.

tag: rip antibody


title: 36--LLC 2wks cancer progression

description: LLC cancer WK2

tag: tissue


title: WT.d4.IN.2 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: WT.d4.IN.1 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: 36b--LLC 1wks cancer progression

description: LLC cancer WK1

value: 7 days


title: WT.d0.IN.1 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: 32-LLC 2wks cancer progression

description: LLC cancer WK2


title: KO.d4.IN.3 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: 57--LLC 3wks cancer progression

description: LLC cancer WK3


title: KO.d0.IN.2 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: WT.d0.IN.2 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: 33--LLC 2wks cancer progression

description: LLC cancer WK2


title: KO.d4.IN.2 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: 31b-LLC 1wks cancer progression

description: LLC cancer WK1


title: KO.d0.IN.3 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: 54-LLC 3wks cancer progression

description: LLC cancer WK3


title: KO.d0.IN.1 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: 38-PBS CONTROL

description: LLC cancer WK0


title: WT.d0.TC.1 (TimeCourse)


title: KO.d4.IN.1 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: WT.d4.IN.3 (RIP_mRNA)

description:
total RNA
pre-AGO2 sample


title: 31-LLC 2wks cancer progression

description: LLC cancer WK2


title: WT.d0.IP.3 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample

value: anti-hAGO2 antibody (a gift from Jay Nelson; Grey et al., 2010)


title: 30b-LLC 1wks cancer progression

description: LLC cancer WK1


title: 30-LLC 2wks cancer progression

description: LLC cancer WK2


title: WT.d0.IP.1 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: 28b-LLC 1wks cancer progression

description: LLC cancer WK1


title: 27-LLC 1wks cancer progression

description: LLC cancer WK1


title: 25--LLC 1wks cancer progression

description: LLC cancer WK1


title: WT.d4.IP.3 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: 23-LLC 1wks cancer progression

description: LLC cancer WK1


title: KO.d0.IP.3 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: WT.d4.IP.2 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: WT.d4.IP.1 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: 21-LLC 1wks cancer progression

description: LLC cancer WK1


title: 2-LLC 4wks cancer progression

description: LLC cancer WK4

value: 28 days


title: KO.d0.IP.2 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: 19-PBS CONTROL

description: LLC cancer WK0


title: KO.d0.IP.1 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: WT.d0.IN.3 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample

data_processing:
smallRNA RIP-Seq reads were pre-processed (includes demultiplexing, barcode trimming, and adaptor removal) using BBDuk from the BBMap toolkit (github.com/BioInfoTools/BBMap, v36.14).
Quality control with FASTQC (bioinformatics.babraham.ac.uk/projects/fastqc/, v0.11.4)
Reads were mapped to the human genome assembly hg38/GRCh38 using Segemehl (v0.2.0-418, Hoffmann et al., 2009). Due to the nature of sRNA-Seq data, multi-mapped reads were considered. For differential expression analysis, only uniquely mapped reads were used.
For differential expression analysis, reads were counted using featureCounts (Liao et al., 2014, version 1.5.2).
Differential expression analysis was performed using DESeq2 (Love et al., 2014) on uniquely mapped reads.
For peak calling, AGO2-IP sequencing data was normalized with the gene expression (pre-IP), which was used to compare relative levels of mRNAs  (separate upload) and miRNAs associated with the RISC complex between WT and ΔmiR-124 cells. We used a custom filtering method to split miRNA read stacks once certain height thresholds were reached. Regions with a summit signal below 10 read pile-ups are considered background and were discarded. With a sliding window approach, starting from the summit, a binding site is first split when its height falls below 20 % of the summit signal. Emerging sub-sites with a summit above this cutoff and 10 pile-ups are then recursively split when their signal falls below 5 % of their summit, which also preserves regions with only weak signal. Replicates of each experimental setup were analyzed separately, and only peaks occurring in all replicates were considered for further analysis. Regions that passed the filtering steps and were called in all replicates were annotated using the ENSEMBL Perl API v90; peaks overlapping miRNA annotation were selected for downstream processing.
Genome_build: hg38
Supplementary_files_format_and_content: RIP_miRNA_Supplement.xlsx: Excel spreadsheet with raw counts from mapping with Segemehl.

extract_protocol:
Cell pellets from approximately 50 million cells were homogenized, lysed in ice-cold lysis buffer (10 mM HEPES [pH 7.3, ThermoFisher Scientific], 100 mM KCl [1049360250, Merck], 0.5 % NP40 [NP40S, Sigma-Aldrich], 5 mM MgCl2, 0.5 mM dithiothreitol [DTT, DTT-RO, Sigma-Aldrich], protease inhibitors [118735, Sigma-Aldrich], recombinant RNase inhibitors [10777019, ThermoFisher Schientific], 1 mM PMSF [36978, ThermoFisher Scientific]) using TissueLyser LT (50 Hz, 2 min, Qiagen). For lysate clearing, homogenates were centrifuged for 15 min at 16,200 × g, 4°C. A 50 µl sample was taken as a pre-IP sample. The RIP samples were incubated with Dynabeads Protein G beads (10009D, ThermoFisher Scientific) coated with anti-hAGO2 antibody (a gift from Jay Nelson; Grey et al., 2010) for 24h at 4°C with end-over-end rotation. After incubation, beads were collected on a Dynamagnet (1 min, on ice, ThermoFisher Scientific) and gently resuspended in low-salt NT2 buffer (50 mM Tris-HCl [pH 7.5, T1503, Sigma-Aldrich], 1 mM MgCl2, 150 mM NaCl [S3014, Sigmal-Aldrich], 0.5 % NP40, 0.5 mM dithiothreitol, 1 mM PMSF, protease inhibitors, recombinant RNase inhibitors). The beads were transferred to a new collection tube and washed with low salt NT2 buffer, followed by two washes with high salt NT2 buffer (50 mM Tris-HCl [pH 7.5], 1 mM MgCl2, 600 mM NaCl, 0.5 % NP40, 0.5 mM DTT, protease inhibitors, 1 mM PMSF, recombinant RNase inhibitors). The solution containing the RNA was then transferred to Qiazol (Qiagen). Subsequently, RNA was isolated from RIP and pre-RIP samples according to the miRNeasy micro kit protocol (Qiagen). The size distribution of the samples and the quality was checked with a Bioanalyzer RNA 6000 Pico Chip (Agilent Genomics).
cDNA libraries were prepared using NEXTFlex Small RNA Library Prep V3 (NOVA-5132-06, BIOO Scientific) with 50 ng input and 22 PCR cycles.


title: 124-PBS CONTROL

description: LLC cancer WK0


title: 125-PBS CONTROL

description: LLC cancer WK0


title: 17-PBS CONTROL

description: LLC cancer WK0


title: 12-LLC 4wks cancer progression

description: LLC cancer WK4


title: WT.d0.IP.2 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: WT.d0.IN.1 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: KO.d4.IP.2 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: KO.d4.IP.3 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: WT.d4.IN.2 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: 111-LLC 3wks cancer progression

description: LLC cancer WK3


title: KO.d4.IP.1 (RIP_mRNA)

description:
AGO2-enriched RNA
post-AGO2 sample


title: 14-PBS CONTROL

description: LLC cancer WK0


title: 107-LLC 4wks cancer progression

description: LLC cancer WK4


title: WT.d0.IN.2 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: 104-LLC 4wks cancer progression

description: LLC cancer WK4


title: 105-LLC 4wks cancer progression

description: LLC cancer WK4


title: 114-LLC 3wks cancer progression

description: LLC cancer WK3


title: WT.d4.IN.3 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: 112-LLC 3wks cancer progression

description: LLC cancer WK3


title: 110-LLC 3wks cancer progression

description: LLC cancer WK3


title: WT.d4.IN.1 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: KO.d0.IN.3 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: WT.d0.IP.3 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: WT.d0.IP.2 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: WT.d0.IP.1 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: WT.d4.IP.3 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: htdp_a315t_fp_29_07_2015

city: Hamburg

first: Giorgio

last: Gonnella

address: Bundesstrasse 43

department: Zentrum für Bioinformatik

institute: Universität Hamburg

description:
Ribosome footprint
Replicate 3- CAGCAT

data_processing:
Library strategy: Ribo-Seq
adapter clipping; cutadapt 1.8.1; parameters: --trimmed-only -e 0
filtering of non-coding RNA; Bowtie2; parameters: seed length: 14, minimum alignment length: 14
splitting of footprint into buckets, according to length; RiboPip; http://github.com/stepf/RiboPip
alignment of footprints to genome; TopHat2; parameters: read mapper: Bowtie2
joining of alignments of each bucket; RiboPip; http://github.com/stepf/RiboPip
count hits per feature; FeatureCounts 1.4.4
differential expression analysis; DeSeq1
Genome_build: GCA_000001635.7
Supplementary_files_format_and_content: tab-delimited files; output of FeatureCounts

source_name: primary cortical neurons

growth_protocol: MN1 cells were grown in D-MEM high glucose GlutaMAX culture medium (Gibco), supplemented with 10% Fetal Bovine serum, 1% Penicillin/Streptomycin antibiotics and 2.4% HEPES. MN1 cells were transiently transfected with plasmids using  Effectene Transfection Reagent (Qiagen) following the manufacturer’s protocol.Neurons were grown on dishes or glass coverslips coated with poly-L-Lysine in Primary Neuro Basal Medium (Lonza) supplemented with NSF-1, 1% Penicillin/Streptomycin antibiotics and L- Glutamine.  0.5 µM Cytosine β-D-arabinofuranoside (Ara-C) was added to the culture on DIV4. Neurons were cultured for 14d at 37°C in a 5% CO2 environment.

extract_protocol: Libraries were generated following original protocol by Ingolia, N.T., et al. (2012) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc, 7, 1534-1550.

treatment_protocol: MN1 cells were treated with 100 µg/mL Cycloheximide (CHX) for 3 mins before lysis.

value: primary cortical neurons

tag: source

value: E16 mouse cortical neurons

tag: stage and strain

value: Harvested DIV14- C57BL/6J

tag: genotype/variation

value: hTDP-43 A315T


title: 11-LLC 4wks cancer progression

description: LLC cancer WK4


title: KO.d0.IP.3 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample

tag: genotype


title: KO.d4.IN.1 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: KO.d4.IN.2 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: KO.d0.IP.1 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: KO.d4.IN.3 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: KO.d0.IN.1 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: KO.d0.IN.2 (RIP_miRNA)

description:
smallRNA
pre-AGO2 sample


title: 10-LLC 4wks cancer progression

description: LLC cancer WK4


title: htdp_a315t_total_29_07_2015

description:
Total mRNA
Replicate 3- ATCAGT

data_processing:
adapter clipping; cutadapt 1.8.1; parameters: --trimmed-only -e 0
filtering of non-coding RNA; Bowtie2; parameters: seed length: 14, minimum alignment length: 14
splitting of footprint into buckets, according to length; RiboPip; http://github.com/stepf/RiboPip
alignment of footprints to genome; TopHat2; parameters: read mapper: Bowtie2
joining of alignments of each bucket; RiboPip; http://github.com/stepf/RiboPip
count hits per feature; FeatureCounts 1.4.4
differential expression analysis; DeSeq1
Genome_build: GCA_000001635.7
Supplementary_files_format_and_content: tab-delimited files; output of FeatureCounts


title: htdp_a315t_fp_16_07_2015

description:
Ribosome footprint
Replicate 2- CAGCAT


title: wt_fp_29_07_2015

description:
Ribosome footprint
Replicate 3-GCCAAT

value: Wild type


title: htdp_wt_total_29_07_2015

description:
Total mRNA
Replicate 3- CTTGTA

value: hTDP-43 WT


title: KO.d4.IP.2 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: WT.d4.IP.2 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: wt_total_29_07_2015

description:
Total mRNA
Replicate 3- ACAGTG


title: WT.d4.IP.1 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: htdp_wt_fp_29_07_2015

description:
Ribosome footprint
Replicate 3- GTGAAA


title: 101-LLC 4wks cancer progression

description: LLC cancer WK4


title: KO.d0.IP.2 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: Protein_footprint_H2O2_Rep3_8hr

city: New York

first: Shuvadeep

last: Maity

state: NY

address: 12 waverly place

department: Center for Genomics and Systems Biology

institute: New york University

description: Protein protected RNA

data_processing:
Library strategy: Protein-bound RNA profiling
illumina output file to fastq file conversion by bcl2fastq version 2.16 and demultiplexing by flexbar version 2.5
Sequenced reads were trimmed for adapter sequence and quality using fastx-toolkit v0.0.14 with parameters -t 20 -l 20 and fastq files from the same sample were merged.
Reads were mapped to abundant sequences to remove ribosomal rDNA, adapter contaminants, PhiX, and polyA/C sequences with bowtie2 v2.2.9 with default settings.
Remaining reads were mapped to hg19 genome with tophat v2.1.1 with parameter --no-coverage-search -G gencode.v19.basic.gtf
Aligned reads were filtered for unique mapping and length: RNA >25, Ribosome footprints 28-30, Protein footprints >25.
Read ounts were generated using htseq v0.6.1p1 with gencode.v19.basic.gtf and parameters --stranded==yes intersection-nonempty.
Genome_build: hg19
Supplementary_files_format_and_content: text file with read counts from Htseq

source_name: cervical cancer cells

growth_protocol: All samples for the total RNA, ribosome footprinting and protein occupancy / footprint profiling were derived from cells grown in parallel. We grew Hela cells under standard conditions, i.e  in DMEM (Sigma) with 10% fetal bovine serum (Atlanta biologicals) and 1X penicillin streptomycin solution (Corning cellgro) at 37℃ and 5% CO2.

extract_protocol:
For RNA seq and ribosome foot print experiment, confluent plates of HeLa cells were first aspirated of their growth media and washed with fresh media supplemented with 0.1 mg/ml cycloheximide. The media was then removed and the cells were washed with 10 ml chilled PBS containing 0.1 mg/ml cycloheximide. Following removal of the PBS, 800 ul of Lysis Buffer was added and the cells were extensively scraped off plate and transferred to a pre-chilled eppendorf tube, recovering ~1 ml of lysate per sample. To insure complete lysis, we passed the lysate through a 25 gauge needle and further incubated on ice for ~10 min. The lysate was then clarified by centrifugation for 10 min at 20,000, 4°C and ∼1 ml supernatant was recovered. For each treatment (tunicamycin and hydrogen peroxide), we aliquoted 100 ul of the cell lysate for total RNA extraction and 200ul for ribosome footprinting. For protein foot print, we scraped crosslinked cells off the plates with a rubber policeman, collected by centrifugation, washed with ice-cold PBS . We resuspended cell pellets in lysis/binding buffer (100 mM Tris-HCl pH 7.5 at 25 °C, 500 mM LiCl, 10 mM EDTA pH 8.0 at 25 °C, 1% LiDS, 5 mM DTT, Complete Mini EDTA-free protease inhibitor (Roche)), incubated them at room temperature for 15 minutes for lysis and passed cells through a 21 gauge needle 10 times for shearing of genomic DNA. Lysates were processed to recover RNA-protein complex as described previously (Munschauer, 2015).
RNA libraries for RNA seq, Ribosome foot print and protein foot print were prepared according to the TruSeq Ribo Profile (Mammalian) kit protocol.

treatment_protocol: At ~60% confluency, we treated the cells with 60 μM H2O2or 0.5 μg/ml tunicamycin  to induce oxidative and ER stress, respectively. We treated samples 8, 4, 1, and 0 hours prior to collection and therefore collected all samples at the same time with similar confluency. For protein occupancy profiling, we added 200 μM of 4-thiouridine (4-SU) at ten hours before the treatment to incorporate photoreactive ribonucleoside analog required for protein occupancy profiling. For ribosome footprinting, we added 0.1 mg/ml cycloheximide for 5 min at 37℃ before the harvesting the cells.

tag: treatment

value: Hydrogen peroxide 8Hr

tag: cell line

value: HeLa


title: Protein_footprint_H2O2_Rep3_4hr

description: Protein protected RNA

value: Hydrogen peroxide 4Hr


title: KO.d4.IP.3 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: htdp_a315t_total_16_07_2015

description:
Total mRNA
Replicate 2- ATCAGT


title: htdp_wt_total_16_07_2015

description:
Total mRNA
Replicate 2- CTTGTA


title: KO.d4.IP.1 (RIP_miRNA)

description:
AGO2-enriched smallRNA
post-AGO2 sample


title: htdp_wt_fp_16_07_2015

description:
Ribosome footprint
Replicate 2- GTGAAA


title: wt_total_16_07_2015

description:
Total mRNA
Replicate 2- ACAGTG


title: wt_fp_16_07_2015

description:
Ribosome footprint
Replicate 2-GCCAAT


title: Protein_footprint_H2O2_Rep3_0hr

description: Protein protected RNA

value: No Hydrogen peroxide


title: hTDP_A315T_neuron_FP_17062015

description:
Ribosome footprint
Replicate 1- CAGCAT


title: Protein_footprint_H2O2_Rep3_1hr

description: Protein protected RNA

value: Hydrogen peroxide 1Hr


title: hTDP_wt_neuron_total_17062015

description:
Total mRNA
Replicate 1- CTTGTA


title: Protein_footprint_H2O2_Rep1_8hr

description: Protein protected RNA


title: Protein_footprint_H2O2_Rep2_1hr

description: Protein protected RNA


title: control_fp_22_05_2015

description:
Ribosome footprint
Replicate 2-GCCAAT

source_name: MN1 cells

value: MN1 cells

value: Clonal hybrid cells of mouse spinal motor neurons and mouse neuroblastoma

value: 10-12 passages

value: GFP transfected control


title: Protein_footprint_H2O2_Rep2_8hr

description: Protein protected RNA


title: htdp_a315t_total_22_05_2015

description:
Total mRNA
Replicate 2- ATCAGT

value: hTDP-43 A315T transfected cells


title: htdp_wt_fp_22_05_2015

description:
Ribosome footprint
Replicate 2- GTGAAA

value: hTDP-43 WT transfected cells