title: MGI08

address: Uppsalalaan 8

institute: Hubrecht Institute

description:
processed data files: RPE-FUCCI_metadata.csv.gz
processed data files: RPE-FUCCI_RPFcounts.csv.gz
processed data files: RPE_MGI_layout.csv.gz

data_processing:
See github repository https://github.com/mvanins/scRiboSeq_manuscript for code and detailed instructions.
Basecalls were performed on the Illumina NextSeq 500. Reads from each plate were demultiplexed using bcl2fastq (version 2.20.0.422) with --use-bases-mask Y*,I*,Y* --no-lane-splitting --mask-short-adapter-reads 0 --minimum-trimmed-read-length 0. Read 1 contains the insert and Read 2 contains the cell barcode.
Next, the UMI was extracted from the first 10 bases of read 1 and concatenated to the start of the cell barcode. Adapter sequences were then trimmed from read 1 using cutadapt (version 2.7) with -m 15: -a TGGAATTCTCGGGT. Trimmed reads were aligned to the reference genome using STARSolo (version 2.7.3a_2020-05-22) with a 50-base overhang (--sjdbOverhang 50) with the following parameters: --seedSearchStartLmax 10 –alignIntronMax 1000000 --outFilterScoreMin 0 --outFilterMultimapNmax 1 --chimScoreSeparation 10 --chimScoreMin 20 --chimSegmentMin 15 --outFilterMismatchNmax 5. Aligned reads were deduplicated with UMI-tools (version 1.0.1) using --spliced-is-unique --per-cell --read-length --no-sort-output, and sorted using sambamba (version 0.7.1). Finally, aligned and deduplicated reads were parsed to extract the mapping coordinates and the reference sequence contexts around both the 5’ and 3’ ends of the reads.
Count tables report the number of unique RPFs aligning within any annotated coding sequence for each gene. Reads were only counted if they aligned to the correct strand, if the trimmed length was between 30 and 45 nt long, and if the trimmed length was equal to the mapped length. Additionally, for these counts, the coding sequence was expanded to include 25 nt upstream and downstream of the start and stop codons, respectively.
A random forest model was trained to predict the A-site location within an RPF read based on the footprint length and the sequence context around the 5’ and 3’ ends. The model was implemented in R (version 3.6.3) using ranger (version 0.12.1) with mlr (version 2.17.1) wrappers for training, tuning, assessment, and prediction. The model was trained on reads spanning a stop codon that satisfied the counting requirements listed above. The number of nucleotides between the 5’ end of these reads and the annotated stop codon was used for training.
hTERT-RPE1-FUCCI cells were only included in downstream analyses if they had more than 4500 unique reads aligning to protein-coding genes, and if more than 85% aligned to coding sequences.
Supplementary_files_format_and_content: Metadata file: Cell-level metadata for cells passing QC. Column Headers: CB: Cell barcode. treatment: Culture/starvation condition prior to sequencing. well: Well location in plate. plate: Plate name. cds: Number of unique reads aligning to coding sequences. utr5: Number of unique reads aligning to 5' UTRs. utr3: Number of unique reads aligning to 3' UTRs. cell_total: Total number of unique reads aligning to protein-coding genes. cds_frac: Fraction of unique total reads aligning to CDS.
Supplementary_files_format_and_content: Count file: Cell-level count tables denoting the number of unique reads aligned to CDS regions for each gene.
Supplementary_files_format_and_content: Plate layout file: Description of the plate layout. Column Headers: well: well location in plate. CB: cell barcode for this well. fraction: Cell fraction/population/treatment.
Genome_build: hg38

source_name: Interphase hTERT RPE1 FUCCI cells

growth_protocol: RPE1 hTERT FUCCI cells (doi: 10.1073/pnas.1322021111) were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), 1× GlutaMAX (Gibco) and 1× Pen-Strep (Gibco) at 37 °C 5% CO2.

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: TCCCGA; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/

treatment_protocol: For the interphase fraction, 7.5E4 cells were plated in a MW-6 and collected by trypsinization (TrypLE, Gibco) 36 hours later. DAPI (ThermoFisher Scientific) was added to cultures as a viability marker, and only viable cells were sorted.

tag: cell type

value: hTERT RPE-1

tag: tissue lineage

value: Retina, eye; pigmented epithelium

tag: genetic tag

value: Fluorescent Ubiquitination-based Cell-Cycle Indicators (FUCCI)

tag: cell fraction

value: Interphase cells

value: Interphase treatment


title: Ground -SH-W3

status: Public on Jun 30 2021

type: RNA

city: Silver Spring

first: Aarti

last: Gautam

state: MD

address: 503 Robert Grant Avenue

department: Medical Readiness Systems Biology

country: USA

institute: WRAIR

description: G-SH-W3 CALLUS

hyb_protocol: cRNA was hybridized to a whole mouse genome microarray (Agilent Technologies) following manufacturer's instructions

scan_protocol: Scanned on a Agilent Technologies Scanner Agilent Technologies G2600D Images were quantified using Agilent Feature Extraction Software v.11.5.1.1 (Agilent Technologies, Inc.).

data_processing: Agilent Feature Extraction Software (v.12.0) was used for background subtraction and LOWESS normalization

molecule: total RNA

organism: Mus musculus

source_name: Callus

label_protocol: Total RNA (200-2000ng) was used as starting material, which was amplified using the Quick Amp Two-Color Kit (Agilent Technologies).The labeled cDNA was purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and eluted, labeled cRNA was quantified using a NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific Inc, Waltham, MA, USA).

growth_protocol: growth protocol is not applicable

extract_protocol: Invitrogen TRIzol RNA extraction methods and the miRNeasy mini kit total RNA purification procedures (QIAGEN Inc., Germantown, MD) were followed to extract the RNA.

tag: strain

tag: age

value: 9-week old

tag: gender

value: Male

tag: sample group

value: Ground

tag: treatment

value: unoperated sham controls

tag: tissue

value: Callus

source_name: Agilent's Universal Mouse Reference RNA composed of total RNA from 11 mouse cell lines

tag: sample type

value: reference


title: Ground -SAL-W4

description: G-SAL-W4 CALLUS

value: segmental bone defect (SBD) surgery


title: MOLM13.GSK862

type: genomic

city: Collegeville

first: Shawn

middle: W

last: Foley

state: PA

address: 1250 S. Collegeville Rd

department: Oncology

institute: GlaxoSmithKline

hyb_protocol: Hybridization was carried out by Q2 Solutions

scan_protocol: Scanning was carried out by Q2 Solutions

data_processing:
Arrays were swan normalized using minfi R package and normalized beta values reported for each sample.
Processed data file includes normalized beta values

label: Cy5 and Cy3

molecule: genomic DNA

organism: Homo sapiens

label_protocol: Labeling was carried out by Q2 Solutions

growth_protocol: Cell lines were obtained from the American Type Culture Collection, the Deutsche Sammlung von Mikroorganismen und Zellbulturen, or AddexBio and maintained in the recommended cell culture media at 37°C in 5%

extract_protocol: Genomic DNA was collected using Zymo Quick gDNA Miniprep and quantified on a nanodrop.

treatment_protocol: Cells were plated 24 hours before treatment with either DMSO or  1 µM GSK3484862 then grown for 96 hours before collection.

tag: cell line


title: GDM1.DMSO

value: DMSO


title: SKM1.GSK862


title: MGI07

type: SRA

city: Utrecht

first: Michael

last: VanInsberghe

state: Utrecht

country: Netherlands

library_source: transcriptomic

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: TATAAT; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/


title: MGI06

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: CTAGCT; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/


title: NOMO1.400nM.GSK032.d6

hyb_protocol: Hybridization was carried out by NYU Medical Center Genome Technology Center

scan_protocol: Scanning was carried out by NYU Medical Center Genome Technology Center

data_processing: Arrays were swan normalized using minfi R package and normalized beta values reported for each sample.

label_protocol: Labeling was carried out by NYU Medical Center Genome Technology Center

extract_protocol: Genomic DNA was collected using Zymo Quick gDNA Miniprep and quantified on a nanodrop

treatment_protocol: Cells were plated 24 hours before treatment with either  DMSO, GSK3484862, GSK3685032, or GSK3510477 then grown for 96 hours before collection.

tag: dose

tag: day


title: MGI05

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: CAGATC; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/


title: NOMO1.0nM.DMSO.d6


title: NOMO1.10000nM.GSK477.d4


title: SKM1.DMSO


title: NOMO1.1000nM.GSK862.d4


title: MGI04

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: TCGAAG; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/


title: NOMO1.2000nM.GSK032.d4


title: Ground -SAL-W1

description: G-SAL-W1 CALLUS


title: NOMO1.80nM.GSK032.d4


title: NOMO1.400nM.GSK032.d4


title: MGI03

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: TCATTC; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/


title: NOMO1.16nM.GSK032.d4


title: NOMO1.3.2nM.GSK032.d4


title: MGI02

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: CTCAGA; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/


title: NOMO1.0nM.DMSO.d4


title: NOMO1.1000nM.GSK862.d2


title: NOMO1.10000nM.GSK477.d2


title: NOMO1.400nM.GSK032.d2


title: MGI01

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
barcode: ATCACG; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/


title: PM Anti-SFPQ

city: Leeds

first: Adrian

last: Whitehouse

address: Leeds

department: 1School of Molecular and Cellular Biology

country: United Kingdom

institute: University of Leeds

data_processing:
Raw reads were quality filtered, and adapter trimmed with Trimmomatic, then aligned to the GRCh38/hg38 assembly of the human genome using HISAT2 (V 2.1.0) with defualt parameters.
Fragments per kilobase of transcript per million mapped reads (FPKM) and transcripts per kilobase (TPM) were calcualted using GRCh38 annotation (GENCODE Release 32) with StringTie (V1.3.4).
Uniquely mapped and non-duplicated reads were extracted for differential peak analysis for SFPQ-IP against IgG-IP.
Peaks were called using MACS2 with parameters -no model, -g 3.0 x 10^8, -p 10^-5.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include coordinates, coverage and abundance estimates (FPKM and TPM) for each sample.
Supplementary_files_format_and_content: bed files include peak calls for SFPQ-IP against IgG control for each cell line.

source_name: PM

growth_protocol: A2058 cells were maintained in DMEM supplemented with 10% foetal bovine serum and 100 U/ml of penicillin and 100 µg/ml of streptomycin. PM were grown in a mixture (2:1) of serum free K-SFM and EMEM. K-SFM was supplemented with 25 µg/ml bovine pituitary extract (BPE), 0.2 ng/ml rEGF, penicillin (100 U/ml), streptomycin (100 µg/ml) and 2 mM L-glutamine (Sigma-Aldrich, UK). EMEM was supplemented with 2% FBS, 1× nonessential amino acids, Primocin (100 U/ml)/streptomycin (100 μg/ml), 2 mM l-glutamine, 5 ng/ml basic fibroblast growth factor and 5 ng/ml endothelin-1.

extract_protocol:
1.0 x 10^6 cells were lysed in RIP Lysis buffer and immunoprecipitated with SFPQ or IgG antibody.
RNA libraries were prepared for sequencing using standard single end and stranded Illumina protocols.

value: Primary melanocytes

tag: rna-ip antibody

value: SFPQ

tag: disease state

value: Normal


title: NOMO1.0nM.DMSO.d2


title: NOMO1.400nM.GSK032.d1


title: PM Anti-IgG

value: IgG


title: THP1.400nM.GSK032.d6


title: NOMO1.10000nM.GSK032.d4


title: NOMO1.0nM.DMSO.d1


title: A2058 Anti-IgG

value: Melanoma cell line A2058

value: Melanoma


title: THP1.10000nM.GSK477.d4


title: THP1.1000nM.GSK862.d4


title: THP1.0nM.DMSO.d6


title: THP1.2000nM.GSK032.d4


title: THP1.10000nM.GSK032.d4


title: THP1.400nM.GSK032.d4


title: 3013807E: whole_blood_Case_56

city: Salvador

first: Artur

middle: Trancoso Lopo

last: Queiroz

address: Rua Waldemar Falcão, 121

country: Brazil

institute: Instituto Gonçalo Moniz

data_processing:
bcl2fastq software (Illumina inc.)
Cutadapt (1.11) was used to extract information of adapter and UMI in raw reads, and output from Cutadapt was used to remove adapter sequences and to collapse reads by UMI with in-house script.
UMI Correction: According to the experiment protocol, each raw read is expected to contain, starting from 5’ end, an insert sequence, the adapter sequence, 12nt-long UMI sequence, and other ligated sequence. Depending on the read length and insert length, not all parts are present in all reads. To correct PCR bias with UMI information, we processed raw reads as described below: 1. Use cutadapt on raw reads with provided adapter sequence to acquire output with information about the presence of adapter for each read. 2. Parse cutadapt output and keep only reads that fulfill all the following requirements: i. Reads contain adapters, ii. Insert sequence should be equal or larger than minimal insert length (default 16nt), iii. UMI sequence should be equal or longer than minimal UMI length (default 10nt). 3. Extract insert sequences from reads which do not contain full length UMI sequence from step 2 output as partial-UMI reads. 4. Examine reads with full length UMI in step 2 output and identify all unique insert+UMI combinations. Extract insert sequences from unique insert + UMI combinations as full-UMI reads. 5. Combine partial-UMI reads and full-UMI reads as output of UMI correction.
Mapping: Bowtie2 (2.2.2) was used for mapping the reads. The mapping criteria for aligning reads to spike-ins, abundant sequence and miRBase are that the reads must have perfect match to the reference sequences. For mapping to the genome, not more than one mismatch is allowed in the first 32 bases of the read. No indels are allowed in mapping.
Supplementary_files_format_and_content: tab-delimited text files include counts values for each Sample and Matrix table with raw miRNA counts for every miRNA and every sample

source_name: whole blood

extract_protocol:
RNA was isolated using the miRNeasy Serum/Plasma Advanced Kit (50) (cat# 217204) according to the manufacturers’ recommendation (QIAGEN).
The library preparation was done using the QIAseq miRNA Library Kit (QIAGEN). A total of 5ul total RNA was converted into miRNA NGS libraries. Adapters containing UMIs were ligated to the RNA. Then RNA was converted to cDNA. The cDNA was amplified using PCR (22 cycles) and during the PCR indices were added. After PCR, the samples were purified. Library preparation QC was performed using either Bioanalyzer 2100 (Agilent) or TapeStation 4200 (Agilent).
Based on quality of the inserts and the concentration measurements the libraries were pooled in equimolar ratios. The library pool(s) were quantified using qPCR. The library pool was then sequenced on a NextSeq500 sequencing instrument according to the manufacturer instructions. Raw data was de-multiplexed and FASTQ files for each sample were generated using the bcl2fastq software (Illumina inc.). FASTQ data were checked using the FastQC tool.

value: HIV-infected

value: whole blood

tag: tb status

value: TB-infected


title: 3013799F: whole_blood_Case_55


title: THP1.0nM.DMSO.d4


title: 3013788I: whole_blood_Case_53


title: A2058 Anti-SFPQ

tag: cell type


title: THP1.10000nM.GSK477.d2


title: 3013786A: whole_blood_Case_52


title: THP1.1000nM.GSK862.d2


title: THP1.400nM.GSK032.d2


title: THP1.0nM.DMSO.d2


title: THP1.400nM.GSK032.d1


title: 3013776B: whole_blood_Case_50


title: 3013785D: whole_blood_Case_51


title: MV411.400nM.GSK032.d6


title: THP1.0nM.DMSO.d1


title: MV411.10000nM.GSK477.d4


title: MV411.1000nM.GSK862.d4


title: MV411.0nM.DMSO.d6


title: 3011850E: whole_blood_Case_49


title: MV411.10000nM.GSK032.d4


title: THP1.80nM.GSK032.d4


title: 3011838B: whole_blood_Case_47


title: 3011837D: whole_blood_Case_46


title: 3013793G: whole_blood_Case_54


title: THP1.3.2nM.GSK032.d4


title: MV411.400nM.GSK032.d4


title: MV411.2000nM.GSK032.d4


title: MV411.80nM.GSK032.d4


title: 3011847C: whole_blood_Case_48


title: MV411.16nM.GSK032.d4


title: 3011835I: whole_blood_Case_45


title: MV411.3.2nM.GSK032.d4


title: THP1.16nM.GSK032.d4


title: 3011824L: whole_blood_Case_44


title: MV411.10000nM.GSK477.d2


title: MV411.0nM.DMSO.d4


title: MV411.1000nM.GSK862.d2


title: 3011823B: whole_blood_Case_43


title: 3011817F: whole_blood_Case_42


title: MV411.400nM.GSK032.d2


title: MV411.400nM.GSK032.d1


title: MV411.0nM.DMSO.d1


title: 3011814A: whole_blood_Case_41


title: 1131408A: whole_blood_Case_40


title: 1111754B: whole_blood_Case_37


title: 1111755L: whole_blood_Case_38


title: 1121051I: whole_blood_Case_39


title: 1111229E: whole_blood_Case_35


title: 1111261F: whole_blood_Case_36


title: Romesco cauliflower curd 2, sample b [ROM-2b]

city: Grenoble

first: Jérémy

last: LUCAS

address: 17 avenue des Martyrs

country: France

institute: CNRS

data_processing:
Sequenced reads were trimmed for adaptor sequence, then mapped to TAIR10 whole genome using STAR with parameters : unique mapping & 6 mismatch maximum
Mapped read were attributed to genes using featureCount (default parameters)
Reads Per Kilobase of gene per Megabase of library size (RPKM) were calculated using a EdgeR from Robinson MD,Bioinformatics. 2010
Genome_build: TAIR10
Supplementary_files_format_and_content: Gene expression (RPKM) per library

molecule: polyA RNA

organism: Brassica oleracea var. botrytis

source_name: Romesco cauliflower curd

growth_protocol: Plants were grown in the field

extract_protocol: RNAs were extracted using the RNeasy Plant Kit protocol from QIAGEN

tag: type

value: Romanesco

value: mature curd tissue


title: 3013814K: whole_blood_Control_33

value: uninfected


title: Romesco cauliflower curd 1, sample 1 [ROM-1]


title: 1111219F: whole_blood_Case_34


title: TREx-BCBL1 RTA_hsa_circ_0001400

city: Baltimore

first: Myong-Hee

last: Sung

address: 251 Bayview Boulevard, Room 06C226

department: National Institute on Aging

institute: NIH

description:
BCBL1.Chimeric.out.junction
BCBL1.back_spliced_junction.bed

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to NC_09333 (Human Herpes virus 8) using STAR with parameters --chimSegmentMin 20 --outFilterMismatchNmax 2. All samples from iSLK-bAC16 (iSLK) or TREx-BCBL1 RTA (BCBL1) are grouped for alignment and downstream processing.
For BCBL1 sample and iSLK sample, Chimeric.out.junction files were used to detect circular RNAs using CIRCexplorer2 parse.
Supplementary_files_format_and_content: bed

source_name: TREx-BCBL1 RTA

growth_protocol: iSLK-BAC16 cells are cultured in DMEM 10% FBS, 1 ug/ml Puromycin, 250 ug/ml G418, 1.2 mg/ml Hygromycin. TREx-BCBL1 RTA cells were cultured in RPMI 10% FBS, 100ug/ml Hygromycin.

extract_protocol:
Total RNA was extracted by Direct-Zol with DNase I digestion
NEBNext Ultra II DNA Library Prep
RT-PCR was performed with specific divergent primers and libraries were prepared from amplicons. Libraries was size-selected to capture 100 to 500 nt of amplicons.

treatment_protocol: Dox (1 ug/ml) and sodium butyrate (1 mM, only for iSLK-BAC16) were added to cell culture for two days.

tag: infection status

value: KSHV positive

value: Dox

value: TREx-BCBL1 RTA


title: Romesco cauliflower curd 2, sample a [ROM-2a]


title: TREx-BCBL1 RTA_kcirc97


title: 3013812C: whole_blood_Control_32


title: 3013804L: whole_blood_Control_31


title: 3013797J: whole_blood_Control_30


title: TREx-BCBL1 RTA_kcircORF72


title: TREx-BCBL1 RTA_kcircT0.7


title: Cauliflower curd 2, sample b [CAUL-2b]

source_name: Cauliflower curd

value: cauliflower


title: 3011859J: whole_blood_Control_28


title: MV411.0nM.DMSO.d2

value: DMSO


title: Cauliflower curd 2, sample a [CAUL-2a]


title: 3011860D: whole_blood_Control_29


title: 3011857B: whole_blood_Control_27


title: Sample15 [204026590072_R07C01]

city: Exeter

first: Jonathan

last: Mill

address: RILD Building, RD&E Hospital

department: Medical School

institute: University of Exeter

description:
204026590072_R07C01_Grn.idat
204026590072_R07C01_Red.idat

hyb_protocol: Samples were processed according to the Illumina Infinium HumanMethylation450K BeadChip (Illumina) protocol.

scan_protocol: Samples were assessed using the Illumina Infinium HumanMethylation450K BeadChip (Illumina) using a Illumina iScan System (Illumina).

data_processing:
Data was imported using the R package methylumi methylumIDAT function. Data was then pre-processed in the R package wateRmelon using the pfilter and dasen functions.
the Nicks_raw_Signal.csv contains unmethylated and methylated signal intensities

label: hapten-labeled dideoxynucleotides

source_name: Human fetal brain

label_protocol: 500 ng DNA from each sample was sodium bisulfite-treated using the EZ-96 DNA Methylation-Gol Kit (Zymo Research) according to the manufacturer’s standard protocol.

extract_protocol: phenol-chloroform extraction

tag: Sex

value: M

tag: age (post-conceptional weeks)

value: fetal brain


title: Cauliflower curd 1, sample 1 [CAUL-1]


title: TREx-BCBL1 RTA_kcirc29


title: iSLK-BAC16_kcirc97

description:
iSLK.Chimeric.out.junction
iSLK.back_spliced_junction.bed

value: Dox+NaB


title: 3011848A: whole_blood_Control_26


title: OBS cauliflower curd sample 6 [OBS-Caul-6]

source_name: OBS cauliflower curd

value: growing curd tissue


title: OBS cauliflower curd sample 5 [OBS-Caul-5]


title: 3011843L: whole_blood_Control_25


title: iSLK-BAC16_kcirc57


title: iSLK-BAC16_kcirc29


title: 3011841D: whole_blood_Control_24


title: OBS cauliflower curd sample 4 [OBS-Caul-4]


title: Human_RNA_4

city: Nice

first: Minoo

last: Rassoulzadegan

address: parc Valrose

department: Institut de Biologie Valrose: iBV

institute: Nice Sophia Antipolis

data_processing:
Library strategy: DRIP
Quality control of the RAW sequencing reads was performed using FastQC version 0.11.5. Trimming of bad-quality reads was performed using Trimmomatic version 0.36 30. Based on the FastQC results we cropped 10 nucleotides from the 5' end of each read, and trimmed bases with Phred score lower than 20 from heading and trailing of each read. Also the Illumina adapter sequences were removed, and the trimmed reads with a size less than 30 bp were removed. Hisat2 was used to align trimmed reads to the reference assembly (GRCh38). HOMER version 4.9 was used for peak finding and generating bedGraph files for visualization of alignment.
Samples were then directly quantified using Salmon v0.12.0 on an index created from the GRCh38 transcript. Then, transcript counts were imported into tximport for all downstream analysis and were then summed by gene symbol.
Genome-wide locations of TERRA repeats were found by aligning sequences of length 24 and 48 composed of 4 and 8 copies of TERRA to the reference genome using bowtie2 using “-a” argument to report all alignments
Genome_build: hg38
Supplementary_files_format_and_content: xlsx file include TPM (Transcript Per Million) values for each Sample

source_name: Sperm

extract_protocol:
Ejaculated human sperm kindly provided by the Fertility Clinics of the University. After sperm preparation TRIzol extracted materials were ethanol precipitated and followed by overnight incubation at 56°C in Tris buffer 20 mM pH8, EDTA 50 mM, with 0.5% SDS, 20 µM dithiothreitol and 400 µg/ml Proteinase K. Two fractions of RNAs were prepared: 1- Free-RNA (R) from aqueous phase of Trizol extraction and, 2- DNAs associated RNAs from interface materials. DNA-bound RNAs were prepared from two sperm samples from man at reproductive age, all showing identical electrophoretic profiles of the DNA-RNA complexes. Both fractions after proteinase K treatment were further purified by the ZR-DuetTM DNA/RNA MiniPrep Plus protocol according to the specifications of the manufacturer.
10-100 ng of RNA were recovered and analysed by high throughput sequencing on Illumina HiSeq 2500 or Illumina MiSeq

value: Sperm

tag: molecule subtype

value: free-RNA


title: TREx-BCBL1 RTA_kcirc57


title: OBS cauliflower curd sample 2 [OBS-Caul-2]


title: Human_RNA_1


title: 3011825J: whole_blood_Control_21

value: HIV-infected


title: Human_D_RNA_1

value: DNA-associated RNA


title: 3011826G: whole_blood_Control_22

value: whole blood


title: Human_D_RNA_4


title: OBS cauliflower curd sample 1 [OBS-Caul-1]


title: 3011816H: whole_blood_Control_20


title: OBS cauliflower curd sample 3 [OBS-Caul-3]


title: 3011815K: whole_blood_Control_19


title: W22_lowN_Rep1

city: New York

first: Chia-Yi

last: Cheng

state: NY

address: 12 Waverly Place

department: Biology

institute: New York University

description: processed data file: NutriNet.maize-leaf.featureCounts.txt

data_processing:
Base calling: (Arabidopsis) Picard Tools version 2.17.11 MINIMUM_QUALITY=2 INCLUDE_NON_PF_READS=false APPLY_EAMSS_FILTER=false. (Maize) bcl2fastq v2.17.1.14 Conversion Software (Illumina).
Removing optical duplicates: Clumpify version 37.24 (Arabidopsis) clumpify.sh -Xmx48g in=in.fq.gz out=out.fq.gz overwrite=t dedupe reorder addcount optical dupedist=40. (Maize) clumpify.sh -Xmx60g in=in.fq.gz out=out.fq.gz overwrite=t dedupe reorder addcount optical dupedist=2500.
Removing adapters: BBDuk version 37.24 (Arabidopsis). (Maize) bbduk.sh in=in.fq.gz out=out.fq.gz overwrite=t ref=~/package/bbmap/resources/adapters.fa ordered=t ktrim=r k=25 mink=11 minlength=35.
Aligning to the genome: BBMap version 37.24 (Arabidopsis). (Maize) bbmap.sh -Xmx56g in=input.fq.gz ordered=t overwrite=t outm=out.sam
Assigning reads: subread v1.5.1 (Arabidopsis) featureCounts -T 1 -s 2 -O -a Araport11.gtf -o out.featureCounts input.sam (Maize) featureCounts -T 1 -R -s 0 -O -a Zea_mays.AGPv4.32.gtf -o out.featureCounts input.sam
Genome_build: (Arabidopsis) TAIR10, (Maize) Zm-B73-REFERENCE-GRAMENE-4.0
Supplementary_files_format_and_content: *.featureCounts.txt: Tab-delimited text files include raw read counts.

organism: Zea mays

source_name: Leaf

extract_protocol:
(Arabidopsis) Total RNA was extracted from leaf samples using Direct-zol MiniPrep by Zymo Research.
(Arabidopsis) NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina.
(Maize) RNAs were isolated with a CTAB, acidified phenol-chloroform extraction, and isopropanol precipitation.
(Maize) TruSeq Stranded mRNAseq Sample Prep kit.

value: Leaf tissue taken two inches from the base of leaf 13, which is the leaf subtending the top ear

tag: age

value: R1 stage

tag: genotype

value: Low nitrogen


title: W22_lowN_Rep3

tag: treatment


title: 3011810I: whole_blood_Control_18

tag: tissue

value: uninfected


title: W22_highN_Rep3

value: High nitrogen


title: 3011809C: whole_blood_Control_17


title: PHG84_lowN_Rep3


title: 3011205L: whole_blood_Control_15


title: VS9-5A

city: Bonn

first: Muhammad Ahmer

last: Jamil

state: NRW

address: Sigmund-Freud Str. 25

department: Institute of experimental haematology and transfusion medicine

country: Germany

institute: University Clinic Bonn

description:
Donor:VS9 Egg:5A
Sample 23

hyb_protocol: done as per Illumina recommendations

scan_protocol: done as per Illumina recommendations

data_processing: Raw data was processed using lumi package of R. Background correction and quantile normalization was done using default parameter settings of lumiExpresso function. Variance stabilization transform was added to lumiExpresso for variance stabilizing. Detection Pvalue was used to filter the probes not expressed in all samples of a cell type

label: biotin

source_name: Cumulus Cells

label_protocol: done as per Illumina recommendations

extract_protocol: Qiagen extraction protcol

treatment_protocol: In vivo ovarian stimulation according to long or short protocol

tag: gender

value: Female

tag: treatment protocol

value: Short

tag: donornum

tag: eggnum

tag: resultcontpreg

value: Neg

tag: resultpregtest


title: 3011839L: whole_blood_Control_23

tag: disease state

tag: tb status


title: 1131413K: whole_blood_Control_13


title: 1121093A: whole_blood_Control_11


title: PHG84_highN_Rep2


title: VS13-5A

description:
Donor:VS13 Egg:5A
Sample 24

value: Long


title: PHG84_highN_Rep1


title: 3011799H: whole_blood_Control_14


title: PHG84_lowN_Rep1


title: VS10-5A

description:
Donor:VS10 Egg:5A
Sample 16

value: Pos


title: VS11-5A

description:
Donor:VS11 Egg:5A
Sample 20


title: PHG84_highN_Rep3


title: VS8-4A

description:
Donor:VS8 Egg:4A
Sample 19


title: 3011806I: whole_blood_Control_16


title: 1121054C: whole_blood_Control_10


title: VS9-3A

description:
Donor:VS9 Egg:3A
Sample 21


title: PH207_lowN_Rep3


title: 1111993H: whole_blood_Control_8


title: PH207_lowN_Rep3-2


title: VS8-2A

description:
Donor:VS8 Egg:2A
Sample 17


title: VS11-1A

description:
Donor:VS11 Egg:1A
Sample 18


title: VS10-4A

description:
Donor:VS10 Egg:4A
Sample 14


title: W22_highN_Rep2


title: PHG84_lowN_Rep2


title: PH207_lowN_Rep2


title: 1111753D: whole_blood_Control_7


title: W22_highN_Rep1


title: PH207_lowN_Rep1


title: VS13-4A

description:
Donor:VS13 Egg:4A
Sample 22


title: 1131374E: whole_blood_Control_12


title: 1111220L: whole_blood_Control_3


title: 1111265J: whole_blood_Control_5


title: PH207_highN_Rep2


title: PH207_highN_Rep1


title: VS6-1A

description:
Donor:VS6 Egg:1A
Sample 10


title: VS6-7A

description:
Donor:VS6 Egg:7A
Sample 13

value: NotUsed


title: VS6-5A

description:
Donor:VS6 Egg:5A
Sample 12


title: 1111213G: whole_blood_Control_2


title: PH207_highN_Rep3


title: 1112000E: whole_blood_Control_9


title: Mo17_lowN_Rep2


title: 1111248E: whole_blood_Control_4


title: 1111476B: whole_blood_Control_6


title: VS2-1A

description:
Donor:VS2 Egg:1A
Sample 9


title: VS2-2A

description:
Donor:VS2 Egg:2A
Sample 11


title: Mo17_lowN_Rep3


title: VS6-9A

description:
Donor:VS6 Egg:9A
Sample 15


title: Mo17_lowN_Rep1


title: UPOP_R1 HeLa

city: Pittsburgh

first: Rajneesh

last: Srivastava

state: Pennsylvania

address: 450 Technology Dr

department: Surgery, University of Pittsburgh

institute: McGowan Institute for Regenerative Medicine

description:
UPOP_R1
Ultra violet crosslinked POP-seq_Replicate_1

data_processing:
POP-seq reads were pre-processed using FASTX toolkits for trimming adaptor sequence ("TGGAATTCTCGGGTGCCAAGG"), and low-quality read fragments. Then the sequence reads were mapped to hg38 (Homo_sapiens.GRCh38.97) reference genome using hisat 0.1.6 with default parameters. Reads mapped onto Human ribosomal DNA complete repeating unit (U13369.1) were discarded.
Aligned reads were post-processed using samtools version 1.2 and formatted for peak calling using bedtools version 2.18.1
Peaks were called using Piranha version 1.2.1 with default parameters
Peaks were merged for the two replicates of each POP-seq variant and used for downstream data analysis
Genome_build: hg38 (Homo_sapiens.GRCh38.97)
Supplementary_files_format_and_content: tsv.gz
Supplementary_files_format_and_content: Merged peak file for the two replicates of NPOP-seq variant
Supplementary_files_format_and_content: Merged peak file for the two replicates of FPOP-seq variant
Supplementary_files_format_and_content: Merged peak file for the two replicates of UPOP-seq variant

source_name: HeLa cells

growth_protocol: HeLa cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s minimal essential medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals) along with 1% antibiotics (penicillin 5000 Units/ml, Streptomycin 5000 μg/ml). All cells were maintained at 37°C and 5% CO2 in a humidified incubator and fresh media was replenished every alternate day until confluent.

extract_protocol:
Following the trizol lysis, interphase pellet was subjected to RNase A/T1 (thermo scientific) degradation in RNase buffer (10 mM Tris-HCl, pH 7.5, 300 mM NaCl and 5 mM EDTA, pH 7.5). 2 µg of RNase A/T1 mix was added to the interphase pellet, mixed by pipetting and incubated at 37° C for 15 min. Interphase-RNase mixture was resolubilized in trizol reagent to recover the RNA-protein complexes. After addition of chloroform, tubes were incubated at room temperature for 5 min, vortexed and centrifuged at 12000 g for 10 min. This results in protein bound RNA at the interphase, unbound RNA in the aqueous phase while the unbound proteins in the organic phase. The aqueous and organic layers were discarded as described previously. Interphase pellet was precipitated in 1 ml methanol and spun down to remove the methanol. Next, the interphase was mixed with Proteinase K in appropriate buffer (0.1M NaCl, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.5% SDS and 200 µg/ml proteinase K). Samples were incubated at 50° C for 2 hours. After proteinase K digestion, free RNA was recovered from the aqueous layer by trizol extraction as described previously.  Samples were cooled and the released RNA was purified by standard trizol extraction. The upper aqueous layer containing the free RNA fragments (previously bound to proteins) was transferred to new tube and precipitated with isopropanol as per the manufacturer instructions.   Protein bound RNA’s concentration was estimated using Nanodrop and up to 1 µg of RNA was incubated with DNase I, 1U (thermo scientific) at 37° C for 30 min to remove any traces of DNA contamination. 1 µl of 50 mM EDTA was added to the reaction mixture and incubated at 65° C for 10 min to terminate the reaction. RNA was purified using conventional trizol extraction from the aqueous layer. At this point, r-RNA depletion was performed with 1 µg input RNA using Ribo-cop kit (Lexogen) as per manufacturer instructions. Further, the ends of r-RNA depleted RNA were modified by treating with Calf intestine alkaline phosphatase (CIAP, Invitrogen) and T4 polynucleotide kinase (T4 PNK, thermo scientific) as per manufacturer protocol.  The end modification enabled the library preparation of these RNA fragments.
RNA purity and concentration were assessed at each step using Nanodrop (thermo scientific) based on the absorbance ratio 260/280 >2. RNA integrity was evaluated using Agilent 2100 bioanalyser system. At least 50 ng of r-RNA depleted POP-seq RNA was used to generate sequencing libraries using the True-seq small RNA library prep kit (Illumina). All libraries were barcoded and sequenced in parallel on a Next-seq platform for 400 million reads to obtain 75 bp single end reads.
POP-seq libraries prepared using Illumina True seq small RNA library preparation kit

treatment_protocol: Cells were cultured in T-175 flask until a maximum of 90% confluency was reached. A total of 100 million cells per replicate of each sample were used for UV, formaldehyde and no-crosslinking control. Cell were washed twice with 1X PBS, the supernatant was removed by pipetting and cells were resuspended in 1X PBS. For UV crosslinking, cells in PBS suspension were transferred to 100 mm dishes and UV irradiated at 254 nm with 400 mJ/cm2 dosage (UV Stratalinker 1800). Immediately after crosslinking, cells were collected in 15 ml tubes and pelleted at 1500 rpm for 5 min. Supernatant was discarded and cells were lysed in 8 ml trizol reagent (Life Technologies) by pipetting up and down to obtain a homogenous cell lysate.    Formaldehyde crosslinking was achieved by pelleting 100 million cells and washing in 1X PBS twice until all the media is discarded. Cells were resuspended in 30 ml PBS in 50 ml tubes and crosslinked with 0.5% formaldehyde (405 µl) for 10 min by gentle shaking at room temperature. Formaldehyde crosslinking was stopped by adding 1 M Glycine (3.75 ml) to the cell suspension for 5 min by gentle shaking at room temperature. Cells were pelleted down at 1500 rpm for 5 min, lysed in 8 ml trizol and homogenized by pipetting up and down. For non-crosslinked samples, cells were pelleted down, washed in 1X PBS and subsequently lysed in trizol for phase separation.

value: Ultra violet crosslinked POP-seq


title: Mo17_highN_Rep3


title: VS1-12A

description:
Donor:VS1 Egg:12A
Sample 7


title: FPOP_R2 HeLa

description:
FPOP_R2
Formaldehyde crosslinked POP-seq_Replicate_2

value: Formaldehyde crosslinked POP-seq


title: UPOP_R2 HeLa

description:
UPOP_R2
Ultra violet crosslinked POP-seq_Replicate_2


title: Mo17_highN_Rep2


title: VS5-8A

description:
Donor:VS5 Egg:8A
Sample 8


title: FPOP_R1 HeLa

description:
FPOP_R1
Formaldehyde crosslinked POP-seq_Replicate_1


title: NPOP_R2 HeLa

description:
NPOP_R2
Non-crosslinked POP-seq_Replicate_2

value: Non-crosslinked POP-seq


title: VS5-5A

description:
Donor:VS5 Egg:5A
Sample 6


title: NPOP_R1 HeLa

description:
NPOP_R1
Non-crosslinked POP-seq_Replicate_1


title: 1111202J: whole_blood_Control_1

value: HIV-infected


title: LH82_lowN_Rep2


title: VS1-10A

description:
Donor:VS1 Egg:10A
Sample 5


title: VS4-8A

description:
Donor:VS4 Egg:8A
Sample 4


title: LH82_highN_Rep3


title: LH82_lowN_Rep1


title: LH82_highN_Rep2


title: LH82_highN_Rep1


title: 1418 - Stroma

city: Boston

first: Andrew

middle: S

last: Liss

state: MA

address: 55 Blossom St

department: Surgery

institute: Massachusetts General Hospital

data_processing:
Paired‐end reads were aligned to the hg38 genome from the University of California, Santa Cruz (http://genome.ucsc.edu) using the STAR version 2.4.0h aligner with default settings (https://github.com/alexdobin/STAR)
Reads that did not map or mapped to multiple locations were discarded. Duplicate reads were marked using the MarkDuplicates tool in picard‐tools‐1.8.4 (Broad Institute, broadinstitute.github.io/picard) and were removed.
The uniquely aligned reads were counted using htseq‐count in the intersection‐strict mode against the Homo_sapiens.GRCh38.79.gtf annotation table from Ensembl (www.ensembl.org).
Prior to analysis, the log2-transformed RNA-seq data was filtered out to exclude poorly expressed genes (defined as a median expression of 0 across all samples) and poorly variant genes (defined as a standard deviation below 0.5).
Genome_build: hg38
Supplementary_files_format_and_content: Tab delineated text files with raw gene counts for every gene and every sample

source_name: PDAC

extract_protocol:
Cancer cells and adjacent stromal cells were microdissected using an Arcturus Pixcell IIE LCM platform and cancer and stromal cells were collected separately on CapSure™ Macro LCM Caps.  RNA was extracted from caputred tissue using a PicoPure™ RNA Isolation Kit.
Amplified cDNA was then generated by using the SMARTer Ultra-Low-input RNA kit, version 4 followed by Nextera® XT DNA Library Preparation kit for sample barcoding and fragmentation per the manufacturer’s protocols.

value: LCM stromal cells

value: PDAC


title: 1548 - Stroma


title: 1656 - Stroma


title: VS4-4A

description:
Donor:VS4 Egg:4A
Sample 2


title: 1385 - Stroma


title: VS1-4A

description:
Donor:VS1 Egg:4A
Sample 1


title: VS1-7A

description:
Donor:VS1 Egg:7A
Sample 3


title: ILP1_lowN_Rep3

value: Leaf tissue taken two inches from the base of leaf 13, which is the leaf subtending the top ear


title: IRHP1_highN_Rep2

value: R1 stage


title: ILP1_lowN_Rep2

value: Low nitrogen


title: 1283 - Stroma


title: 1310 - Stroma


title: LH82_lowN_Rep3


title: IRHP1_lowN_Rep1


title: 1106 - Stroma


title: 1184 - Stroma


title: ILP1_highN_Rep2


title: Yellow_diet_replicate_3

city: PADOVA

first: GIUSEPPE

last: PERSICO

state: PD

address: VIA UGO BASSI 58/B

country: Italy

institute: UNIVERSITA' DI PADOVA

library_source: genomic

data_processing:
Reads were aligned to mm10 using ‘bwa’
Unmapped reads, reads with a MAPQ smaller then 1, duplicate reads and those that mapped outside of chr 1-19 and Chr X were removed using ‘samtools
Peak calling was done with SICER2 software using the following parameters: fragment size=200, window size=200, gap size= 400 and FDR< 0.05
Genome_build: mm10
Supplementary_files_format_and_content: Binarized file produced by bwa containing sequence alignment data.
Supplementary_files_format_and_content: Text file containing enriched regions coordinates

source_name: Liver

extract_protocol:
Chromatin extraction was performed starting from four section of formalin fixed paraffin-embedded (FFPE) tissues.
Libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Mice were sacrificed by cervical dislocation and livers were washed in phosphate buffer (PBS) and incubated overnight at room temperature (RT) in 4% formalin solution. Formalin-fixed samples were then dehydrated by increasing concentration of ethanol (from 70% to 100%) and subsequently included in paraffin.

tag: diet

value: yellow corn-based ad libitum

tag: strain

value: 12 month

value: Liver

tag: antibody


title: Yellow_diet_replicate_2


title: IHP1_lowN_Rep3


title: Yellow_diet_replicate_1


title: 984 - Stroma


title: 916 - Stroma


title: IHP1_lowN_Rep2


title: IHP1_lowN_Rep1


title: Standard_diet_replicate_3

value: standard diet ad libitum


title: Standard_diet_replicate_2


title: 911 - Stroma


title: IHP1_highN_Rep3

value: High nitrogen


title: IHP1_highN_Rep2


title: Standard_diet_replicate_1


title: 895 - Stroma


title: Red_diet_replicate_3

value: purple corn-based ad libitum


title: FR1064_lowN_Rep1


title: IHP1_highN_Rep1


title: Red_diet_replicate_1


title: B73xPHG84_lowN_Rep3


title: Red_diet_replicate_2


title: 864 - Stroma


title: 855 - Stroma


title: 891 - Stroma


title: B73xPHG84_lowN_Rep1


title: 818 - Stroma


title: 1039 - Stroma


title: High_fat_replicate_2

value: high fat-diet ad libitum


title: ILP1_highN_Rep1


title: 933 - Stroma


title: 1656 - Tumor

value: LCM cancer cells


title: High_fat_replicate_3


title: B73xPHG84_highN_Rep3


title: Caloric_restriction_replicate_3

value: Caloric restriction


title: B73xPHG47_lowN_Rep3


title: 1548 - Tumor


title: Caloric_restriction_replicate_2


title: B73xPHG47_lowN_Rep2


title: 1418 - Tumor


title: Caloric_restriction_replicate_1


title: 1385 - Tumor


title: B73xPHG47_highN_Rep3


title: 1310 - Tumor


title: B73xPHG47_highN_Rep2


title: 1283 - Tumor


title: B73xPHG47_highN_Rep1


title: 1106 - Tumor


title: 1184 - Tumor


title: B73xPH207_lowN_Rep3


title: B73xPH207_lowN_Rep1


title: B73xPH207_lowN_Rep2


title: 754 - Stroma


title: High_fat_replicate_1


title: B73xPH207_highN_Rep3


title: B73xPH207_highN_Rep2


title: B73xPHG84_highN_Rep1


title: B73xPH207_highN_Rep1


title: 911 - Tumor


title: B73xOh7B_lowN_Rep3


title: 891 - Tumor


title: 933 - Tumor


title: 916 - Tumor


title: 895 - Tumor


title: B73xOh7B_lowN_Rep1


title: MC38.ms023_1uM.I

city: Seattle

first: James

middle: Douglas

last: Thomas

state: WA

address: 1100 Fairview Ave. N., B3-111

department: Computational Biology

institute: Fred Hutchinson Cancer Research Center

description: RNA-seq of cell line treated with the indicated compound

data_processing:
Illumina Casava software was used for base calling.
For RNA-seq, reads were aligned to the mouse transcriptome using RSEM followed by mapping to the mouse genome (mm10) using Bowtie and TopHat.
RNA splicing was quantified using MISO.
Genome_build: mm10 for mouse data
Supplementary_files_format_and_content: The final processed data is found in the file "d.psi.txt"

extract_protocol:
RNA was extracted from cells of interest using an RNeasy kit (Qiagen, Frederick, MD) and quantified using a NanoDrop 8000 (ThermoFisher Scientific); a minimum of 500 ng of high quality RNA (as determined by Agilent Bioanalyzer) per sample or replicate was used for library preparation.
Poly(A)-selected, stranded Illumina libraries were prepared with a modified TruSeq protocol and sequenced on the Illumina HiSeq 2000

treatment_protocol: Cell lines were treated with splicing inhibitors at the indicated concentrations for 96 hours in vitro, harvested and washed three times with PBS in excess to remove all drug, and then used for downstream analyses, continued growth in vitro or tumor challenge in vivo into syngeneic animals.

tag: cell line


title: MC38.ms023_1uM.H


title: B73xOh7B_highN_Rep2


title: B73xOh7B_highN_Rep3


title: 855 - Tumor


title: B73xOh7B_highN_Rep1


title: 984 - Tumor


title: 1039 - Tumor


title: B73xMo18W_lowN_Rep1


title: 818 - Tumor


title: 754 - Tumor


title: Control3 (1 µM DOX)

city: Shreveport

first: Tarek Magdy

last: Mohamed

state: LA

address: 1501 Kings Highway, BRI F3-15/22

department: Pathology

institute: LSU Health Shreveport

data_processing:
Reads were mapped to the GRCh38 reference human genome using Subread software (Liao et al., 2013). Gene expression levels and exon usage were estimated using featureCounts function in the Subread software (Liao et al., 2013). Differential gene expression analysis was done using DEseq2 package (Love et al., 2014) and R (v3.3.3).
Genome_build: GRCh38
Supplementary_files_format_and_content: raw counts

source_name: hiPSC-CMs

growth_protocol: Cardiomyocytes were diffrentiated and maintained according to our establisshed protocol (Burridge et al., 2014)

extract_protocol:
RNA was extracted using TRI Reagent and Direct–zol RNA microprep kit (both Zymo) including on-column DNase digestion to remove genomic DNA. Samples were quantified using an Agilent 2100 Bioanalyzer and passed QC.
Library preparation was done using a TruSeq RNA v2 kit (Illumina) and sequencing with NextSeq 500 instrument (Illumina) by Northwestern’s NuSeq core facility, generating ~40 million single-end 75 bp reads for each sample

treatment_protocol: Day 30 hiPSC–CMs from 6 patients (3 Control and 3 S427L) were treated with 1 mM doxorubicin or vehicle for 24 h

tag: dox-induced toxicity

value: NO

value: 1 µM DOX

tag: rs2229774 genotype

value: GG


title: B73xMo18W_lowN_Rep2

tag: age

tag: genotype


title: MC38.indisulam_1uM.C


title: MC38.indisulam_1uM.B


title: MC38.dmso.I


title: B73xMo18W_highN_Rep3

tag: tissue

tag: treatment


title: Control2 (1 µM DOX)


title: MC38.ms023_1uM.G


title: MC38.indisulam_1uM.A


title: B73xMo18W_highN_Rep2


title: MC38.dmso.H


title: 864 - Tumor


title: Control1 (1 µM DOX)


title: MC38.dmso.G


title: MC38.dmso.C


title: S417L2 (1 µM DOX)

value: YES

value: GA


title: B73xMo18W_highN_Rep1

value: Leaf tissue taken two inches from the base of leaf 13, which is the leaf subtending the top ear


title: B73xMo17_highN_Rep3

value: R1 stage


title: B73xMo17_lowN_Rep2

value: Low nitrogen


title: MC38.dmso.B


title: S417L1 (1 µM DOX)


title: B73xMo17_highN_Rep2


title: Control3 (0 µM DOX)


title: MB49.ms023_1uM.I


title: B73xLH82_lowN_Rep3


title: MC38.dmso.A


title: MB49.ms023_1uM.H


title: B73xMo17_highN_Rep1


title: Control1 (0 µM DOX)


title: MB49.ms023_1uM.G


title: S417L3 (0 µM DOX)


title: Control2 (0 µM DOX)


title: B73xLH82_highN_Rep3


title: MB49.indisulam_1uM.C


title: MB49.indisulam_1uM.B


title: B73xLH82_lowN_Rep2


title: B73xLH82_highN_Rep2


title: S417L3 (1 µM DOX)


title: B73xLH82_highN_Rep1


title: S417L2 (0 µM DOX)


title: B73xILP1_lowN_Rep3


title: S417L1 (0 µM DOX)


title: MB49.dmso.I


title: MB49.indisulam_1uM.A


title: B73xILP1_lowN_Rep1


title: MB49.dmso.G


title: B73xILP1_highN_Rep3


title: D_Mtb

first: Monika

last: Looney

address: 1503 E Jefferson Street

institute: Johns Hopkins University

data_processing:
read alighment using HISAT2
sorting and SAM/BAM conversion using samtools
read assembly, quantification, merging using Stringtie
transcript abundance estimation using Ballgown
differential expression analysis using prepDE.py and DESeq2
Genome_build: GRCh38_latest_genomic.fna
Supplementary_files_format_and_content: CSV files containing gene or transcript count matrix

source_name: Primary monocyte-derived macrophages

growth_protocol: Monocytes were extracted from platelet-depleted blood from healthy human donors by Ficoll density gradient centrifugation and plastic adherence. Monocytes were allowed to adhere for 4 hours, after which non-adherent non-monocytes were washed away in 1X phosphate-buffered saline. Monocytes were differentiated for one week. Cells were cultured in RPMI-1640 + 10% fetal bovine serum + 4mM L-glutamine.

extract_protocol:
Total RNA was isolated using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Cat # 80204) following manufacutrer instructions.
RNA sequencing libraries were prepared using the Illumina TruSeq RNA library prep kit v2 (Illumina, Cat # RS-122-2011)

treatment_protocol: After 1 week differentiation, macrophages were either left uninfected or infected with Mtb strain H37Rv at a MOI of 10 for 48 hours.

tag: cell type

value: Primary monocyte-derived macrophages

tag: donor

value: D

tag: moi


title: B73xILP1_highN_Rep2


title: B_Mtb

value: B


title: C_Mtb

value: C


title: A_Mtb

value: A


title: MB49.dmso.C


title: D_uninfected

value: uninfected


title: MB49.dmso.B


title: B73xIHP1_lowN_Rep3


title: B73xILP1_highN_Rep1

value: High nitrogen


title: C_uninfected


title: B73xIHP1_highN_Rep3


title: CT26.ms023_1uM.I


title: A_uninfected


title: MB49.dmso.A


title: B73xIHP1_highN_Rep2


title: CT26.ms023_1uM.H


title: SR206

address: Uppsalalaan 8

institute: Hubrecht Institute

description:
processed_data_file: RPE-FUCCI_metadata.csv.gz
processed_data_file: RPE-FUCCI_RPFcounts.csv.gz
processed_data_file: RPE_SR2_layout.csv.gz

data_processing:
See github repository https://github.com/mvanins/scRiboSeq_manuscript for code and detailed instructions.
Basecalls were performed on the Illumina NextSeq 500. Reads from each plate were demultiplexed using bcl2fastq (version 2.20.0.422) with --use-bases-mask Y*,I*,Y* --no-lane-splitting --mask-short-adapter-reads 0 --minimum-trimmed-read-length 0. Read 1 contains the insert and Read 2 contains the cell barcode.
Next, the UMI was extracted from the first 10 bases of read 1 and concatenated to the start of the cell barcode. Adapter sequences were then trimmed from read 1 using cutadapt (version 2.7) with -m 15: -a TGGAATTCTCGGGT. Trimmed reads were aligned to the reference genome using STARSolo (version 2.7.3a_2020-05-22) with a 50-base overhang (--sjdbOverhang 50) with the following parameters: --seedSearchStartLmax 10 –alignIntronMax 1000000 --outFilterScoreMin 0 --outFilterMultimapNmax 1 --chimScoreSeparation 10 --chimScoreMin 20 --chimSegmentMin 15 --outFilterMismatchNmax 5. Aligned reads were deduplicated with UMI-tools (version 1.0.1) using --spliced-is-unique --per-cell --read-length --no-sort-output, and sorted using sambamba (version 0.7.1). Finally, aligned and deduplicated reads were parsed to extract the mapping coordinates and the reference sequence contexts around both the 5’ and 3’ ends of the reads.
Count tables report the number of unique RPFs aligning within any annotated coding sequence for each gene. Reads were only counted if they aligned to the correct strand, if the trimmed length was between 30 and 45 nt long, and if the trimmed length was equal to the mapped length. Additionally, for these counts, the coding sequence was expanded to include 25 nt upstream and downstream of the start and stop codons, respectively.
A random forest model was trained to predict the A-site location within an RPF read based on the footprint length and the sequence context around the 5’ and 3’ ends. The model was implemented in R (version 3.6.3) using ranger (version 0.12.1) with mlr (version 2.17.1) wrappers for training, tuning, assessment, and prediction. The model was trained on reads spanning a stop codon that satisfied the counting requirements listed above. The number of nucleotides between the 5’ end of these reads and the annotated stop codon was used for training.
hTERT-RPE1-FUCCI cells were only included in downstream analyses if they had more than 4500 unique reads aligning to protein-coding genes, and if more than 85% aligned to coding sequences.
Supplementary_files_format_and_content: Metadata file: Cell-level metadata for cells passing QC. Column Headers: CB: Cell barcode. treatment: Culture/starvation condition prior to sequencing. well: Well location in plate. plate: Plate name. cds: Number of unique reads aligning to coding sequences. utr5: Number of unique reads aligning to 5' UTRs. utr3: Number of unique reads aligning to 3' UTRs. cell_total: Total number of unique reads aligning to protein-coding genes. cds_frac: Fraction of unique total reads aligning to CDS.
Supplementary_files_format_and_content: Count file: Cell-level count tables denoting the number of unique reads aligned to CDS regions for each gene.
Supplementary_files_format_and_content: Plate layout file: Description of the plate layout. Column Headers: well: well location in plate. CB: cell barcode for this well. fraction: Cell fraction/population/treatment.
Genome_build: hg38

source_name: Interphase hTERT RPE1 FUCCI cells

extract_protocol:
Single cells were index sorted using a BD FACS Influx into 384-well hardshell plates (BioRad) that were pre-filled with 5 µL of light mineral oil (Sigma Aldrich) and 50 nL of lysis buffer [22 mM Tris-HCl pH 7.5, 16.5 mM MgCl2, 5.5 mM CaCl2, 165 mM NaCl, 1.1 % Triton X-100, 2.2 U/µL RNaseIN Plus (Promega), 0.11 mg/mL Cyclohexamide (Sigma Aldrich)]. After sorting, plates were spun down at 2000 ×g for 2 minutes and kept on wet ice until all plates were ready for further processing. No RNA purification was performed.
To generate ribosome-protected footprints, 50 nL of Micrococcal Nuclease (MNase, 10500 U/µL, New England Biolabs) was added to each well, and plates were incubated at 37 °C for 30 minutes. In order to stop digestion, 50 nL of stop mix [0.0186 U/µL Thermolabile Proteinase K (New England Biolabs), 62 mM EGTA (Sigma Aldrich), 16.5 mM EDTA (Ambion), and 697.5 mM guanidium thiocyanite (GuSCN, Sigma Aldrich)] was added to each well, and plates were incubated at 37 °C for 30 minutes then 55 °C for 10 minutes and held at 4 °C.
After ribosome footprint digestion, libraries were constructed using a one-pot small-RNA library preparation protocol that incorporated end repair, two RNA ligations, cDNA synthesis, and an indexing PCR. First, 50 nL of end-repair mix [4.1× of 10× T4 RNA Ligase Buffer (New England Biolabs), 16.4 mM MgCl2, 4.1 mM uridine triphosphate (New England Biolabs), 1.37 U/µL T4  Polynucleotide Kinase (New England Biolabs), and 0.82 U/µL RNaseIN Plus] was added to each well, and plates were incubated at 37 °C for 1 hour and held at 4 °C. Next, 264 nL of 3’ ligation brew [1× T4 RNA Ligase Buffer (New England Biolabs), 1 µM pre-adenylated 3’ adapter (OMV630 Integrated DNA Technologies), 35.5 % PEG-8000 (New England Biolabs), 0.1 % Tween-20 (Sigma Aldrich), 1 U/µL RNaseIN Plus, and 21.3 U/µL T4 RNA Ligase 2 Truncated KQ (New England Biolabs)] was added to each well and plates were incubated at 4 °C for 18 hours. The cDNA synthesis primer was then pre-annealed to the 3’ ligation products by adding 50 nL of the RT primer mix [5.2 µM RT primer (OMV572, Integrated DNA Technologies), 13.5 µM adenosine triphosphate (ATP, New England Biolabs), and 1 % Tween-20] to each well, heating to 65 °C for 1 minute, 37 °C for 2 minutes, 25 °C for 2 minutes, and holding at 4 °C. Five-prime adapters were then ligated by adding 156 nL of 5’ ligation brew [1× T4 RNA Ligase Buffer, 30.75 % PEG-8000, 0.1 % Tween-20, 0.5 µM 5’ adapter (OMV632, Integrated DNA Technologies), 1.25 U/µL T4 RNA Ligase 1 (Ambion)] and incubating at 37 °C for 2 hours and holding at 4 °C. Complementary DNA synthesis was then peformed by adding 771 nL of reverse transcription brew [1.88× 5× RT Buffer (ThermoFisher Scientific), 1.25 mM dNTPs (Promega), 0.1875 % Tween-20, 1.875 U/µL RNaseIN Plus, and 9.375 U/µL Maxima H Minus Reverse Transcriptase (ThermoFisher Scientific)] to each well, and heating at 50 °C for 1 hour, then 85 °C for 5 minutes and holding at 4 °C. Finally, single-cell libraries were indexed during PCR by first transferring 150 nL of 20 µM unique forward index primers (Integrated DNA Technologies) and 3.2 µL of PCR brew [1.5× Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs), 0.15 % Tween-20, and 0.94 µM reverse index primer (Integrated DNA Technologies)] to each well. Plates were then incubated at 98 °C for 30 s followed by 10 cycles of 98 °C for 15 s, 65 °C 30 s, 72 °C for 30 s, and then a final incubation at 72 °C for 5 min and holding at 4 °C. Plates were then frozen at -20 °C until pooling.
After library construction the plates were pooled and purified. The contents of each plate were first collected in VBLOK200 reservoirs (Click Bio) by centrifuging at 2000 ×g for 2 min. The aqueous phase (~1.9 mL per plate) was separated from the light mineral oil by centrifugation, and concentrated to approximately 500 µL using n-butanol (Sigma Aldrich) and diethyl ether (Sigma Aldrich). Product was then cleaned up using AMPure XP beads (Beckman Coulter) that had been diluted 5× in bead binding buffer [20 % PEG-8000 (Sigma Aldrich) 2.5 M Sodium Chloride (Sigma Aldrich)]; diluted beads were added to the sample at a 2.1:1 ratio, and the final product was resuspended in 50 µL of low TE buffer [LoTET, 3 mM Tris-HCl pH 8.0 (Ambion), 0.2 mM EDTA pH 8.0 (Gibco), 0.1 % Tween-20]. Half of each of the cleaned-up library pools was then run on a 10 cm 7 % polyacrylamide gel at 200 V for ~6 h, and the ~10 base-pair region from 175-185, corresponding to an insert size of ~30-40 nt was excised. The band was then crushed and soaked overnight at 4 °C in elution buffer [5:1 LoTET:7.5 M ammonium acetate (Sigma Aldrich)]. Finally, eluate was precipitated in ethanol.
adapters: barcode: ACTTGA; 5' Adapter: rCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUr(N1:25252525)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1)r(N1); 3' Adapter: /5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/

value: hTERT RPE-1

tag: tissue lineage

value: Retina, eye; pigmented epithelium

tag: genetic tag

value: Fluorescent Ubiquitination-based Cell-Cycle Indicators (FUCCI)

tag: cell fraction

value: Interphase cells

value: Interphase treatment


title: CT26.indisulam_1uM.C


title: B73xIHP1_highN_Rep1


title: MB49.dmso.H