title: NIC, HEK293, in vivo (SSII)

source_name: Total RNA from HEK293 cells, in vivo treated with NIC


title: 2A3, HEK293, in vivo (SSII)

status: Public on Dec 16 2020

type: SRA

city: Groningen

first: Danny

last: Incarnato

state: Netherlands

address: Nijenborgh 7

department: Molecular Genetics

country: Netherlands

institute: University of Groningen

library_source: transcriptomic

data_processing:
Library strategy: SHAPE-MaP
All the relevant data analysis steps have been conducted using RNA Framework v2.6.9 (PMID: 29893890; http://www.rnaframework.com). Reads pre-processing and mapping was performed using the rf-map module (parameters: -ctn -cmn 0 -cqo -cq5 20 -b2 -mp "--very-sensitive-local"). Reads were trimmed of terminal Ns and low-quality bases (Phred < 20). Reads with internal Ns were discarded. Mapping was performed using the “very-sensitive-local” preset of Bowtie2. The mutational signal was derived using the rf-count module (parameters: -m -rd), enabling the right re-alignment of deletions. Grid search of optimal slope/intercept pairs was performed using the rf-jackknife module (parameters: -rp “-md 600 -nlp” -x) and ViennaRNA package v2.4.11, by disallowing isolated base-pairs, setting the maximum base-pairing distance to 600 nt and enabling the relaxed structure comparison mode (a basepair i/j is considered to be correctly predicted if any of the following pairs exist in the reference structure: i/j; i-1/j; i+1/j; i/j-1; i/j+1).
Supplementary_files_format_and_content: Additional data files (RC/XML files for analysis with RNA Framework [http://www.rnaframework.com]) are available at: http://www.incarnatolab.com/datasets/2A3_Marinus_2020.php

molecule: total RNA

organism: Homo sapiens

source_name: Total RNA from HEK293 cells, in vivo treated with 2A3

extract_protocol:
Cells pellets were lysed by direct addition of 1 mL ice-cold QIAzol, followed by a brief incubation at 56°C for 5 min to completely dissolve cell aggregates. 200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min.
Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer [65 mM Tris-HCl pH 8.0; 95 mM KCl; 4 mM MgCl2], then purified with NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 µl (for SSII and HIV RT) or 2.5 µl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 µl 20 µM random hexamers and either 0.25 µl (for SSII) or 0.5 µl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 µl. For HIV RT, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 40 mM MgCl2], 0.5 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT (Worthington Biochemical Corporation, cat. HIVRT). For TGIRT-III, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], 0.25 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT (InGex, cat. TGIRT-50). For SSII, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 0.5 µl DTT 0.1 M, 0.25 µl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III), or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 µg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 µl of a 1:2 dilution of protease inhibitor cocktail (Sigma Aldrich, cat. P9599) in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111L). Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), following manufacturer instructions.

treatment_protocol: HEK293 cells were grown in DMEM (4.5 g/L D-Glucose), supplemented with 10% heat-inactivated FBS, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 U/mL penicillin and 25 μg/mL streptomycin, at 37°C (5% CO2), to ~75% confluence. Cells were then covered with a thin layer of Trypsin-EDTA (0.25%) solution, incubated at room temperature for 1 min, then dissociated by pipetting up and down with complete medium. Cells were centrifuged at 180 x g for 1 min and medium discarded. Cells were then resuspended in 450 µl 1X PBS (pH 7.4) and 50 µl of each SHAPE compound were added, followed by gentle mixing by tapping the tube. Cells were allowed to react at 37°C for 15 min, with moderate shaking, after which 500 µl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 10,000 x g for 1 min (4°C) and supernatant discarded.

tag: strain


title: WT m6A-IP rep 1

city: Chicago

first: Allen

last: Zhu

state: Illinois

address: 929 E 57th St, Room E331

country: USA

institute: University of Chicago

description: m6A IP poly A RNA

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were first mapped to the mycoplasma genome to check for contamination and remove any reads (parameter -k 7), then mapped to the mm10 whole genome using Hisat2 v2.1.0 with parameters -k 1
For m6A-seq, our lab's software packages RADAR (https://scottzijiezhang.github.io/RADARmanual/Mannual.html) and MeRIPtools (https://scottzijiezhang.github.io/MeRIPtoolsManual/index.html) were used to call peaks. QNB was used to perform differential methylation analysis.
For RNA-seq, we used the input samples only. DESeq2 was used to count reads for each gene and to perform differential expression analysis.
Genome_build: mm10
Supplementary_files_format_and_content: DEG-all.csv: DESeq2
Supplementary_files_format_and_content: allPeakResults.csv: MeRIPtools
Supplementary_files_format_and_content: m6A-diff-Me-Peaks.txt: QNB

molecule: polyA RNA

organism: Mus musculus

source_name: Mouse Embryonic Stem Cells

growth_protocol: The R1 ESCs were cultured on mitomycin C-treated mouse embryonic fibroblasts in ES medium containing DMEM supplemented with 15% FBS, 1 mM L-glutamine, 0.1 mM mercaptoethanol, 1% non-essential amino acid , 1% Pen/Strep, nucleosides 1,000 U /ml leukemia inhibitory factor, 3mM CHIR99021 and 1mM PD0325901.

extract_protocol:
Cells and RNA was harvested using Trizol reagent. Total RNA was extracted and underwent one round of poly-A Dynabeads purification (ThermoFisher). RNA was then fragmented, and Input saved. Fragmented RNA then underwent m6A-immunoprecipitation. Illumina TruSeq v2 RNA Sample Prep Kit (Cat#20020595) was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols
m6A MeRIP-seq

value: R1 cell line

tag: cell type

value: embryonic stem cell

tag: tissue

value: inner cell mass

tag: genotype

value: Mettl3/Wtap KD; R-WT METTL3/WTAP


title: ZR751shLucif_rna_rep2

city: Nashville

first: Thomas

middle: Paul

last: Stricker

state: TN

address: 1161 21st Ave South, CC3309A

department: Pathology, Microbiology and Immunology

institute: Vanderbilt University

description:
RNAseq
3040-TPS-215
RNAseq_fpkm_table

data_processing:
FastQC v.0.11.4
trimming fastq-mcf, ea-utils, v1.1.2
alignmment Tophat (v2.0.13)
quantified using cufflinks (v2.2.1)
normalized using cuffnorm (v2.2.1)
Genome_build: hg19
Supplementary_files_format_and_content: RNAseq_fpkm_table

source_name: ZR75-1 cell line

growth_protocol: ZR751 cells (RRID CVCL_0588) were obtained from the Lannigan laboratory41 and grown in RPMI (Sigma Aldrich #R8758500ml) supplemented with 10% heat-inactivated FBS (Corning™ #35016CV), 0.002% insulin (Sigma Aldrich #11376497001) and 50 IU penicillin, 50 mg/mL streptomycin (Corning™ #MT30001CI). The cell culture incubator parameters were as follows: 37 ˚C, 95% relative humidity, and 5% CO2 concentration.

extract_protocol:
Cells were harvested at steady-state using the RNAEasy Kit (Qiagen 74104). ChIPs were performed for two biological replicates, for one experimental repetition. Cells were grown to 80% confluency, washed 3 times in ice-cold PBS (8g NaCl, 0.2g KCl, 1.44g Na2HP04, 0.24gKH2PO4, H2O up to 1L, adjusted to pH 7.4 with HCl) and then fixed for 10 minutes at room temperature using 7% formaldehyde (50ml cold PBS, 2.5ml formaldehyde Sigma Aldrich 252549), followed by quenching with 2.5 ml of 2.5M glycine (93.8g glycine Sigma Aldrich G7126 in 500ml H2O) for two minutes at room temperature. After aspirating and washing with 50 ml cold PBS, we lysed the cells using 20ml Farnham lysis buffer (5mM HEPES pH 8.0, 85mM KCl, 0.5% NP-40) and 400ul protease inhibitor cocktail (PIC, Roche 11873580001) in order to scrape the cells off (Corning™ 3008) into a 50ml conical tube (Corning 352098). These tubes were spun down at 2,000 rpm for five minutes at 4° Celsius.     Nuclei lysis buffer (50mM Tris-HCl ph8, 10mM EDTA pH8, 1% SDS), 1X PIC, and 10mM sodium butyrate (Sigma Aldrich B5887) were added to a concentration of ~2e7 cells per 400 ul and resuspended until homogenous. Chromatin was sonicated using a Covaris LE220 for 35 minutes, then centrifuged at max speed for 10 minutes at 4°C to obtain supernatant. Per 0.1 ml of supernatant, we diluted with 0.9ml ChIP dilution buffer (50mM Tris-HCl pH 8, 0.167M NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate), 0.5ml RIPA-150 (50mM Tris-HCl pH8, 0.15M NaCl, 1mM EDTA pH8, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate), 28ul 50X PIC, and 14ul 1M sodium butyrate.      Anti-ERα (3ul/IP), anti-H3K4me1 (1ul/IP), and anti-SP1 (3ul/IP) were linked to 100ul/IP, 60ul/IP, and 100ul/IP magnetic anti-rabbit Dynabeads respectively with RIPA-150 up to 500ul for 6 hours at 4°C for 6 hours in low-bind tubes (Eppendorf Z666505), and then incubated with 150ug of chromatin overnight at 4°C. Immunoprecipitants were washed with RIPA-150, RIPA-500 (50mM Tris-HCl pH8, 0.5M NaCl, 1mM EDTA pH8, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate) twice, RIPA-LiCl (50mM Tris-HCl pH8, 1mM EDTA pH8, 1% Nonidet P-40, 0.7% sodium deoxycholate, 0.5M LiCl2) twice, and 1X TE Buffer pH8 (10mM Tris-HCl pH8, 1mM EDTA pH8) twice for 5 minutes each. Chromatin-IPs were eluted from the beads in 200 ul freshly made Direct Elution Buffer (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA pH8, 0.5% SDS), and then treated with 1ul 1mg/ml RNase A (Fisher Scientific FEREN0531) at 65°C with shaking for 4 hours to reverse crosslinks. This was followed by 3ul proteinase-K (Sigma-Aldrich 3115879001) overnight at 55°C. DNA was purified using phenol–chloroform extraction. Samples were transferred to a spun-down 2ml phase lock gel tube (Qiagen 129056) and an equal volume of phenol/chloroform/isoamyl alcohol (Sigma Aldrich P3803100ML) was added. This was spun at room temperature for 5 minutes at 14,000g, and the sample was moved to a new 1.5ml tube.  One tenth volume sodium acetate (Invitrogen AM9740), 1ul glycogen (Roche 10901393001), twice volume 100% ethanol (Sigma Aldrich E7023500ML) was added, and the samples were incubated at -80°C for 30 minutes. The sample was spun at max speed for 30 minutes at 4°C, and the supernatant was carefully aspirated. The pellet was washed with 1ml cold 70% ethanol, and spun at max speed for 30 minutes at 4°C. The supernatant was aspirated, and the spin was repeated a final time. The supernatant was removed, and pellet was allowed to dry. The pellet was then resuspended in 25ul elution buffer (Qiagen 19086) and subsequently quantified by Qubit 2.0 Fluorometer.
Standard Illumina ChIP-seq Library Kits (IP-202-1012, IP-202-1024) were used to build sequencing libraries for two biological replicates per condition for one experimental repetition, with inputs used as control. . RNA quality was assessed in VANTAGE via Invitrogen Qubit and Agilent BioAnalyzer and samples with RIN >7 were used. RNA libraries were generated with two biological replicates of 2 μg RNA using Illumina’s TruSeq Stranded Total RNA Sample Prep Kit (20020597).

treatment_protocol: Oligos to use for shRNA were designed and ordered from Sigma/Genosys at the Molecular Cell Biology Core at Vanderbilt. The oligos were annealed, phosphorylated, and ligated into pSuper42–44 for transformation into DH5α cells. QIAprep Spin Miniprep Kit (Qiagen 27104) was used to isolate the vector, which was transfected into ZR751 cells and assessed by qPCR for KD.  KDs that worked were then isolated with QIAprep Spin Miniprep Kit (Qiagen 27104), digested, and ligated into pLVTH45 (Addgene 12262) for transformation into STBL3 cells. A QIAGEN Plasmid Plus Maxi Kit (Qiagen 12963) isolated the pLVTH for transfection into 293T cells, from which lentivirus was collected. The oligo sequence used to silence MLL3 was 5° - CCGGCGCACCTTATAGTAAACAGTTCTCGAGAACTGTTTACTATAAGGTGCGTTTTT -3°, taken from The RNAi Consortium.46 Negative control Luciferase shRNA Control was donated by the Lannigan laboratory.47 Cells were stably transduced at 100,000 cells per well in a 6-well plate (Corning 3516) with 4 ul lentivirus, and subsequently flow sorted for GFP expression and propidium iodide (Sigma Aldrich P4864) staining after 3 days. qPCR was performed in technical triplicate to check shRNA KD 3 days after transduction. Experiments were performed in multiple, but early (<=10) passages of the stably transduced cell lines. The antibodies used for ChIP-seq were anti-ERα (Santa Cruz Biotechnology sc-543X), anti-H3K4me1 (Abcam ab8895), anti-SP1 (Abcam ab13370), and sheep anti-rabbit IgG Dynabeads M-280 (Invitrogen™ 11203D).

tag: cell line

value: breast cancer cell line ZR75-1

value: shLucif/control


title: 3A2A input rep 2

description: polyA RNA

value: Mettl3/Wtap KD; R-3A2A METTL3/WTAP


title: MX4

city: Pullman

first: Michael

middle: K

last: Skinner

state: WA

address: Abelson 507

department: SBS

institute: WSU

description: multiple.dmr.results.csv.gz

library_source: genomic

data_processing:
Basic read quality was verified using summaries produced by FastQC. The raw reads were trimmed and filtered using Trimmomatic. The reads for each MeDIP sample were mapped to the Rnor 6.0 rat genome using Bowtie2 with default parameter options. The mapped read files were then converted to sorted BAM files using SAMtools.
To identify DMRs, the reference genome was broken into 1000 bp windows. Genomic windows with a mean of less than 10 mapped reads per sample were removed prior to further analysis. The MEDIPS R package was then used to calculate differential coverage between disease and non-disease sample groups. The edgeR p-value was used to determine the relative difference between the two groups for each genomic window.
Windows with an edgeR p-value less than an arbitrarily selected threshold were considered DMRs. The DMR edges were extended until no genomic window with a p-value less than 0.1 remained within 1000 bp of the DMR.
CpG density and other information was then calculated for the DMR based on the reference genome.
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: The results of the edgeR analysis in CSV format. This includes raw read counts for each genomic window as well as the calculated p-value and other summary values.

molecule: genomic DNA

organism: Rattus norvegicus

source_name: Sperm

growth_protocol: Female and male rats of an outbred strain Hsd:Sprague Dawley SD (Harlan) at 70 to 100 days of age were fed ad lib with a standard rat diet and ad lib tap water. The gestating female rats treated were designated as the F0 generation. F1- F3 generation control and  methoxychlor lineages were housed in the same room and racks with lighting, food and water. All experimental protocols for the procedures with rats were pre-approved by the Washington State University Animal Care and Use Committee (protocol IACUC # 6252).

extract_protocol:
For each sample, the epididymis was dissected free of fat and connective tissue, then, after cutting open the cauda, placed into 6 ml of phosphate buffer saline (PBS) for 20 minutes at room temperature. Further incubation at 4ºC will immobilize the sperm.  The tissue was then minced, the released sperm pelleted at 4ºC 3,000 x g for 10 min, then resuspended in NIM buffer and stored at -80ºC for further processing.   DNA protocol: While sperm are thawing, turn on the centrifuge to 4ºC.   1. Re-suspend sperm pellet with pestle (and pestle motor if available).   o If there is some liquid left with the pellet, there is no need to add any additional buffer  o If not add 100 ?l 1xPBS or NIM buffer  2. Take 25 to 50 ?l of the suspension (depending on how big the pellet is) and transfer it to a microfuge tube with 200 ?l 1xPBS.   o Mix  3. Sonicate suspension at 25% with probe sonicator for 5 seconds.  4. Spin at 12,000 rpm for 10 minutes at 4?C.  o While waiting, turn on heat plate to 65?C   5. Discard supernatant. Be careful – pellet is loose.  6. Wash with 200 ?l 1xPBS and spin as above.  o While waiting, retrieve DTT from freezer (make sure there is enough)    o Turn on the incubator  o Vortex DTT once it has thawed a bit  7. Resuspend pellet in 100 ?l 1xPBS, then continue with protocol below.  General:  1. Re-suspend tissue in 100 ?l PBS or NIM buffer, then   o add 820 ?l DNA extraction buffer   o add 50 ?l 0.1M DTT   o vortex briefly  o incubate at 65?C for 15 minutes   o Retrieve ProteinaseK from freezer  2. Add 50 ?l proteinase K (20 mg/ml) and   o incubate at 55?C for 2-3 hours under constant rotation  o use Eppendorf SafeLock tubes and wrap the tops with Parafilm  3. After incubation add 300 ?l of protein precipitation solution (Promega Genomic DNA Purification Kit, A795A)  o mix (vortex) thoroughly and make sure that the solutions are mixed   o then incubate for 20 min on ice (or longer if needed)  4. Centrifuge at 13,500 g for 30 minutes at 4?C.  o Prepare 2 mL tubes  o Take glycoblue out of the freezer  5. Transfer 1 ml of the supernatant to a new 2 ml tube.   o add 2 ?l of glycoblue   o add 1 ml of cold 100 % isopropanol  6. Mix well by inverting tubes several times.  7. Incubate for 60 min at -20?C.  8. Centrifuge for 20 min at 13,500 g at 4?C.   9. Take off and discard supernatant without disturbing (blue) pellet.   10. Wash pellet 2-3 times with 70% cold ethanol.   o Add 500?l of 70% ethanol to each pellet and   o return to freezer for 20 minutes  11. Centrifuge for 10 min at 4?C at maximum speed (18,200 g)   o discard supernatant  12. Spin again briefly to collect residual ethanol at bottom of tube and remove as much liquid as possible with gel loading tip.  13. Air-dry at RT until pellet looks dry, but be careful not to over-dry.   o This will take about 5 minutes  14. Re-suspend in 100 ?l of nuclease free water or TE.  15. Measure DNA concentration using Nanodrop.   Methylated DNA immunoprecipitation protocol: Shear & Add Antibody to DNA  1. Sonicate purified genomic DNA using Covaris:  a. Dilute 6?g genomic DNA into 50 or 130 ?l TE buffer in the appropriate Covaris tube.  b. Set Covaris to desired program (300 or 500 bp program works well).  c. Run program for each tube  • While waiting, turn on the dry bath to 95ºC.   2. Run 10 ?l sonicated DNA & 5 ?l DNA ladder on 2% agarose gel to verify fragment size.  • While waiting for gel, clean up Covaris.   3. Measure volume of sonicated DNA after gel run.  4. Dilute sonicated DNA with TE buffer to 400 ?l (400 – 130 + 10 = 280 µL)  5. Heat-denature for 10min at 95?C, and   • immediately cool on (watery) ice for 5min  6. To the denatured sonicated DNA:  a. Add 100?l of cold 5X IP buffer   b. Add 5?g (volume depending on concentration) of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006). ALWAYS check antibody concentration. Diagenode sometimes changes it.  c. Incubate the DNA-antibody mixture overnight on a rotator at 4?C.  Rotate at a low enough speed to prevent significant foaming.  Bind Beads to DNA: Antibody Mixture  1. Pre-wash Magnetic Beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) as follows:  • Resuspend the beads in the vial (pipet up and down or rotate, do not vortex)  • Transfer desired volume (50 ?l per sample) to a centrifuge tube, add the same volume of Washing Buffer (at least 1 mL), and resuspend.  • Place the tube in a magnet for 1-2 minutes and discard supernatant.   • Resuspend beads in 1 ml of washing buffer and incubate for 1 minute on bench. Put tube on magnet for 1-2 minutes and discard supernatant.  • Remove the tube from the magnet and resuspend the washed beads in the same volume of 1xIP buffer as the initial volume of beads.  2. Add 50?l of beads to the 500?l of DNA-antibody mixture from step 2  3. Incubate 2h on a rotating platform at 4?C.  Wash DNA: Antibody: Beads Mixture & Add Proteinase K Mix  1. Wash beads three times with 1X IP buffer as follows:  • Place tube in magnet for 1-2 minutes and discard supernatant  • Remove tube from magnet  • Add 1ml cold 1X IP buffer  • Mix by inverting tube or gently vortexing (set Vortex to low).  • Incubate tube(s) for 1 minute on bench.  • Place tube in magnet for 1-2 minutes and discard supernatant  • Repeat twice for a total of 3 washes  2. Resuspend the beads in 250?l digestion buffer.  3. Add 3.5?l Proteinase K Mix (20mg/ml) to the resuspended beads.  4. Incubate 2-3 hours on a rotating platform at 55?C (make sure to seal lids so they don’t leak). Use USA Scientific SealRite tubes or Eppendorf Safe-Lock tubes and seal top with Parafilm.  Purify DNA  • Retrieve Glycoblue from freezer.   1. Remove Parafilm and add 250?l Phenol-Chloroform-Isoamylalcohol to each tube.    • Vortex for 30 sec and centrifuge at 14,000g for 5min at RT.  • Remove the aqueous supernatant and transfer to a fresh microcentrifuge tube.  2. Add 250?l chloroform to the supernatant from step 1.    • Vortex for 30sec and centrifuge at 14,000g for 5min at RT.   • Remove the aqueous supernatant and transfer to a fresh microcentrifuge tube.  3. Add 500 ?l ethanol  • Then add 20?l 5M NaCl   • Then add 2?l glyco-blue (20mg/ml)   • Vortex well   4. Precipitate in -20?C freezer for 1 hour to overnight.  5. Centrifuge at 14,000g for 20min at 4?C.  Carefully remove the supernatant, while not disturbing the pellet.  6. Wash the pellet with 500?l cold 75% ethanol. Leave in -20?C freezer for 15 min.  7. Centrifuge at 14,000g for 5min at 4?C.  Carefully remove the supernatant.  8. Completely dry samples.  9. Resuspend in 25?l H2O.    10. Measure the DNA concentration in Qubit with ssDNA kit and matching buffer.
MeDIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra DNA Library following the manufacturer’s protocol  and indexing each sample individually with NEBNext Multiplex Oligos for Illumina. NEBNext Ultra II Library Prep Kit for Illumina: ssDNA  • Start with 20 µl in PCR strips.   o ~40 ng of DNA  o Complete the volume to 20 µl   • Add 2.5 µL of diluted (1:10) random primer to each tube.   o for 10 samples, 27 µL 0.1x TE and 3 µL primers  o for 12 samples 36 µL 0.1x TE and 4 µL primers  o for 22 samples, 54 µL 0.1x TE and 6 µL primers  • Place strips in the thermocycler and use the Denaturation program. Immediately remove (as soon as time runs out on the program) and cool on watery ice following denaturation. Let tubes rest on watery ice for 5 minutes, then transfer to refrigerated block.   I. Second Strand cDNA Synthesis  Assemble the second strand cDNA synthesis reaction on ice by adding the following components to the 20 µl DNA.   Second Strand Synthesis Reaction  DNA          20 µL  ORANGE NEBNet 2nd Strand Synthesis Reaction Buffer  8 µL  ORANGE NEBNext 2nd Strand Synthesis Enzyme Mix 4 µL  Nuclease-free water      45.5 µL  Total Volume        ~70 µL  1. Keeping the tube on ice, mix thoroughly by pipetting t

treatment_protocol: Outbred Sprague Dawley gestating female rats (F0 generation) were administered an intraperitoneal dose of 200 mg/kg body weight of methoxychlor. These doses were administered at 90 days of age, during embryonic days 8-14 (E8-E14) of fetal gonadal sex determination. The F1 generation offspring was directly exposed as a fetus and F2 generation grand-offspring exposed as the germline in the F1 generation. These were each bred at 90 days of age within the lineage. The F3 generation great-grand-offspring is required to establish the transgenerational inheritance generation of ancestral exposure.

tag: measurement

value: MeDIP

tag: disease

value: multiple disease

tag: lineage

value: methoxychlor


title: 3A2A input rep 3

description: polyA RNA


title: 6A3, HEK293, in vivo (SSII)

source_name: Total RNA from HEK293 cells, in vivo treated with 6A3


title: 3A2A input rep 1

description: polyA RNA


title: MX2

description: multiple.dmr.results.csv.gz


title: WT m6A-IP rep 2

description: m6A IP poly A RNA


title: ZR751shLucif_Input

description:
ChipSeq input control
2501-KS-13

data_processing:
FastQC v.0.11.4
trimming fastq-mcf, ea-utils, v1.1.2
alignment bwa version 0.7.5a-r405
Post-alignment filtering Samtools 1.752 and Picard 1.126 MarkDuplicates.
Quality, enrichment PhantomPeakQualTools v1.2.153
Peak calling SPP v1.15.5
Genome_build: hg19
Supplementary_files_format_and_content: per sample peak files

tag: chip antibody


title: WT input rep 2

description: polyA RNA


title: ZR751shMLL3_Input

description:
ChipSeq input control
3040-TPS-118


title: WT input rep 3

description: polyA RNA


title: MX1

description: prostate.dmr.results.csv.gz

value: prostate


title: I5, HEK293, in vivo (SSII)

source_name: Total RNA from HEK293 cells, in vivo treated with I5


title: 1M4, HEK293, in vivo (SSII)

source_name: Total RNA from HEK293 cells, in vivo treated with 1M4


title: ZR751shLucif_SP1_rep2

description:
SP1 ChipSeq
2501-KS-7

value: anti-SP1 (Abcam ab13370)


title: WT input rep 1

description: polyA RNA


title: ZR751shLucif_SP1_rep1

description:
SP1 ChipSeq
2501-KS-6


title: DMSO, HEK293, in vivo (SSII)

source_name: Total RNA from HEK293 cells, in vivo treated with DMSO


title: NAI, HEK293, in vivo (SSII)

source_name: Total RNA from HEK293 cells, in vivo treated with NAI


title: MM3040_72h

city: Pittsburgh

first: Timothy

last: Billiar

state: Pennsylvania

address: 200 Lothrop Street

department: Surgery

institute: University of Pittsburgh

description:
All time points for the same patient were processed in the same batch.
MM3040_aggr.txt.gz

data_processing:
To minimize the potential batch effect, we analyzed each batch of mouse data separately. For each batch, raw sequencing data was processed using the 10x CellRanger pipeline, cellranger count followed by cellranger aggr (--normalize=mapped, mouse: cell ranger v2.1.0, mapped to mm10) to generate a UMI count matrix and then further processed using the Seurat (v2.3.4). Genes expressed in ≥ 3 cells were retained. Cells with gene counts less than 200 or more than 5000 were filtered out. The UMI count matrixes, which pass the above preliminary filtering and contain the data collected from all the samples from the same batch (01-04 or 11-14) or paralel batches (18-23) , are provided as the processed data.
Based on the mouse experiments, biological replicates prepared in parallel were highly reproducible. Thus, similar to the mouse analysis, different time points sampled from the same patients with the matched healthy control were processed by cellranger count/aggr (human: cell ranger v3.0.0, mapped to GRCh38, aggr: --normalize=mapped) and then by Seurat (v3.0.2) for quality control and pre-processing. Genes expressed in ≥ 3 cells were retained. Cells with gene counts less than 200 or more than 5000 or ≥ 20% mitochondria genes were filtered out. The UMI count matrixes, which passed the above preliminary filtering and contained the data collected from 3 time points and corresponding control for each patient, are provided as the processed data. To overcome human heterogeneity and to identify the same cell type or functional state in population, the data from different individuals were integrated by Seurat integration standard workflow.
Genome_build: Mouse: mm10. Human: GRCh38.
Supplementary_files_format_and_content: For the samples processed in the same batch (samples for each patient, mouse 01-04 or mouse 11-14) or parallel batches (mouse 18-23), the UMI count matrixes which were generated by cellranger aggr (--normalize=mapped) and passed Seurat preliminary filtering are provided.

source_name: PBMC

extract_protocol:
Ficoll centrifugation to isolate PBMC or BMMC.
RNA libraries were prepared for sequencing using 10x Genomic single cell gene expression procotol: mouse (v2) and human (v3).

value: PBMC

tag: group


title: ZR751shMLL3_SP1_rep2

description:
SP1 ChipSeq
2501-KS-15


title: STR_YAYC_10

city: Niigata

first: Tomoyuki

last: Yamanaka

address: 1-757 Asahimachidori, Chuo-ku

department: Brain Research Institute

country: Japan

institute: Niigata University

data_processing:
Total 11 samples were analyzed in two sequencing lanes and finally 24~37M reads (mean qualities > 37) were obtained for each sample. Obtained seq data were analyzed using Agilent Strand NGS to identify DEGs by NF-Y knockdown. Genes with lower expressions (max of the read counts is less than 200) were excluded.
Genome_build: mm9
Supplementary_files_format_and_content: A tab-delimited text file for DEGs (P < 0.05, 20-100 percentile)

source_name: FACS-sorted striatum cells

extract_protocol:
Total RNAs were prepared from the sorted cells by RNeasy Micro Kit (Qiagen), and analyzed by Agilent Bionalyzer. Their RNA integrity numbers (RINs) were 6.7~9.0 (average is 8.2) and obvious RNA degradation was not observed.
Because the RNA concentrations were low (~2 ng / μL), we used Clontech SMARTer Ultra Low Input RNA Kit for Sequencing (v3) for efficient amplification of cDNA after reverse transcription according to the manufacture's protocol. Libraries for sequencing were prepared by Nextera XT Sample Prep Kit, and sequenced by an Illumina HiSeq 2500 system (single read; 50 cycles; rapid run mode).

tag: treatment

value: AAV-EmGFP-miR-YAYC

value: FACS-sorted striatum cells


title: STR_YAYC_22


title: ZR751shMLL3_SP1_rep1

description:
SP1 ChipSeq
2501-KS-8


title: MM3040_4h


title: MM3040_24h


title: STR_YAYC_8


title: ZR751shLucif_ERa_rep2

description:
ERa ChipSeq
2501-KS-4

value: anti-ERalpha(Santa Cruz Biotechnology sc-543X)


title: MM3040_Ctrl

value: Ctrl


title: STR_YAYC_4


title: Jag1 mutant_rep2

type: RNA

city: Baltimore

first: Angelika

last: Doetzlhofer

state: MD

address: 855 North Wolfe St.

department: Neuroscience

institute: The Johns Hopkins University School of Medicine

description: tamoxifen/progesterone

hyb_protocol: 17 hours at 45° C with rotation (60rpm) as described by Affymetrix in their GeneChip Expression Analysis Technical Manual.

scan_protocol: Using Affymetrix’ GeneChip Scanner 3000 7G and default parameters described by the manufacturer in their GeneChip Expression Analysis Technical Manual.

data_processing:
Affymetrix CEL files were extracted with the Partek GS 6.6 platform, and their data RMA and quantile-normalized to create log2 transcript signal values, which were used in subsequent t-test ANOVA analysis.
MTA-1_0.r3.pgf
MTA-1_0.na36.mm10.probeset.csv

label: biotin

source_name: Sox2-CreER/+ Jag1 fx/fx_cochlear epithelium

label_protocol: RNA was labeled with the Affymetrix GeneChip WT Pico Reagent Kit using the manufacturer's protocol.

growth_protocol: Sox2-CreER/+ Jag1 fx/fx male were timed mated to Jag1 fx/fx female to produce stage E14.5 Sox2-CreER/+ Jag1 fx/fx (experimental) and  Jag1 fx/fx (control) embryos

extract_protocol: miRNeasy Micro Kit (Qiagen) was used to extract total RNA from individual cochlear epithelia preparations

treatment_protocol: Timed-pregnant dam received tamoxifen and progesterone to induce Jag1 deletion at stage E14.5. Five days later, inner ear cochleas were harvested from stage P0 Sox2-CreER/+ Jag1 fx/fx (Jag1 mut) and Jag1 fx/fx (Jag1 wt) animals. After removing the caspule, spiral ganglion and cochlear roof, the remaining cochlear tissue was incubated in a 1:1 mix of collagenase and dspase to purify the cochlear epithelia.

tag: dev stage

value: stage P0

value: Jag1 mutant


title: Jag1 mutant_rep3

description: tamoxifen/progesterone


title: Jag1 wild type_rep3

description: tamoxifen/progesterone

source_name: Jag1 fx/fx_cochlear epithelium

value: Jag1 wild type


title: A4-10_S29

city: Faro

first: Elvira

last: Fatsini

address: Universidade do Algarve

country: Portugal

institute: UALG

data_processing:
Adapter removal: Cutadapt 2.1
Mapping: Bowtie2 v2.4.1
Annotation: Htseq-count 0.6.0

organism: Solea senegalensis

source_name: plasma sample

extract_protocol:
SncRNAs were isolated from blood plasma samples using miRNeasy Serum/Plasma Kit (Qiagen, Germany) following manufacturer instructions, and assessment of RNA quality and quantity was performed in a 2200 TapeStation Nucleic Acid system using High Sensitivity RNA ScreenTapes (Agilent, USA). Library preparation of 30 samples were performed using NEXTflex Small RNA-Seq kit v3 (Bioo Scientific, USA) for Illumina platforms following manufacturer protocol, and libraries size, purity, and concentration were evaluated on a High Sensitivity D1000 ScreenTape (Agilent, USA). Normalized libraries were clustered in one pool and multiplexed sequencing was done in two independent runs (15 libraries per run) by the NextSeq500 sequencer using a NextSeq High Output kit v2 (75 cycles) (Illumina, San Diego, CA), following the protocol provided by the manufacturer.
NEXTflex Small RNA-Seq kit v3 (Bioo Scientific, USA)

treatment_protocol: The fish maintained in two of the four tanks (n = 31 fish) were muscularly injected with KISS2 decapeptide at a dose of 250 µg/kg body weight, based on previous reports of positive results of KISS2-10 eliciting gonadotropin release and gonadal development. The remaining fish (n = 31) were injected with phosphate buffered saline (PBS) to test the placebo effect and thus to be used as a control group. The same day, blood samples were collected 2 (2h) and 4 (4h) hours after treatment, as previously described, to determine the acute effect on hormonal levels. Further samplings were performed 2 (2d) and 4 (4d) days after the treatments, to collect blood. At this stage, also plasma was collected for detection of microRNAs. For that, samples of blood (up to 2 mL) were collected from each fish and deposited in a 2 mL Eppendorf tube previously treated with cold buffered sodium citrate (3.8 % in 0.01 M PBS), as well as the syringe. After 15 min at 4 °C, plasma was separated from other blood components through differential centrifugation (15 min at 3000 g) and collected. No visible sign of haemolysis was noted in collected plasma samples. A total of 100 μL of plasma was sampled to perform sex hormone quantification and stored at -20 °C until steroid extraction. The remaining plasma was re-centrifuged at 3000 g for 5 min (to avoid cell debris contamination) and 500 μL of supernatant plasma was collected, snap-frozen in liquid nitrogen, and stored at -80 °C until RNA isolation and analysis.

tag: Sex

value: Male

value: Plasma

value: Kiss treated

tag: sampling point


title: Jag1 wild type_rep2

description: tamoxifen/progesterone


title: Jag1 mutant_rep1

description: tamoxifen/progesterone


title: A4-17_S30


title: MM3038_24h

description:
All time points for the same patient were processed in the same batch.
MM3038_aggr.txt.gz


title: STR_YAYC_6


title: STR_NT2_21

value: AAV-EmGFP-miR-NT2 injection


title: DKST-1-1

city: seoul

first: Kyung Bae

last: Min

address: yonsei-ro 50-1

department: college of medicine

country: South Korea

institute: Yonsei University

data_processing:
RNA quality was assessed by an Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using an ND-2000 Spectrophotometer (Thermo Inc., DE, USA). For control and testing RNAs, rRNA was removed using a Ribo-Zero Magnetic kit (Epicentre Inc., USA) from each 5 ug of total RNA. The construction of a library was performed using a SMARTer Stranded RNA-Seq Kit (Clontech Lab Inc., CA, USA) according to the manufacturer’s instructions. The rRNA depleted RNAs were used for the cDNA synthesis and shearing, following manufacturer’s instructions. Indexing was performed using the Illumina indexes 1–12. The enrichment step was carried out using PCR.
libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a StepOne Real-Time PCR System (Life Technologies Inc., USA). High throughput sequencing was performed as paired-end 100 sequencing using HiSeq 2500 (Illumina Inc., USA). Bacterial-Seq reads were mapped using a Bowtie2 software tool to obtain the alignment file.
Differentially expressed genes were determined based on counts from unique and multiple alignments using EdgeR within R (R development Core Team, 2016) using Bioconductor (45). The alignment file also was used for assembling transcripts. The quantile normalization method was used for comparisons between samples. Gene classification was based on searches done by DAVID (http://david.abcc.ncifcrf.gov/).

organism: Pseudomonas aeruginosa

source_name: DKST-1_bacterial total RNA

growth_protocol: P. aeruginosa PAO1 was incubated for 2 h in 5 ml of LB with vigorous shaking at 37°C. Then, additional incubation was conducted for 1 h under the same condition with 150 𝜇M of Dkstatin-1 or Dkstatin-2 supplement.

extract_protocol:
Total RNA samples were extracted using an RNeasy mini kit (Qiagen), and total DNA was eliminated using RNase-free DNase set (Qiagen) following the manufacturer’s instructions. The extraction was performed as an experimental duplicate.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: wild type

value: 150 µM of Dkstatin-1 supplement


title: MM3038_Ctrl


title: ZR751shLucif_ERa_rep1

description:
ERa ChipSeq
3040-TPS-137

value: breast cancer cell line ZR75-1

value: shLucif/control


title: MM3038_72h


title: A4-9_S28


title: ZR751shMLL3_ERa_rep1

description:
ERa ChipSeq
3040-TPS-124


title: ZR751shMLL3_ERa_rep2

description:
ERa ChipSeq
3040-TPS-135


title: ZR751shLucif_H3K4me1_rep1

description:
H3K4me1 ChipSeq
3040-TPS-126

value: anti-H3K4me1 (Abcam ab8895)


title: STR_NT2_23


title: STR_NT2_5


title: MM3020_72h

description:
All time points for the same patient were processed in the same batch.
MM3020_aggr.txt.gz

value: PBMC


title: ZR751shLucif_H3K4me1_rep2

description:
H3K4me1 ChipSeq
3040-TPS-121


title: A4-8_S27


title: A4-3_S26


title: MM3038_4h


title: ZR751shMLL3_H3K4me1_rep2

description:
H3K4me1 ChipSeq
3040-TPS-119


title: A3-6_S23

value: Untreated


title: STR_NT2_9

value: FACS-sorted striatum cells


title: A3-11_S25


title: MM3020_4h


title: A3-5_S22


title: ZR751shMLL3_H3K4me1_rep1

description:
H3K4me1 ChipSeq
3040-TPS-125


title: MM3020_Ctrl


title: MM3015_Ctrl

description:
All time points for the same patient were processed in the same batch.
MM3015_aggr.txt.gz


title: KIN-CLIP Sample 16_1mW laser 1x Dazl 0sec

city: Cleveland

first: Deepak

last: Sharma

state: Ohio

address: 10900, Euclid Avenue

department: Center for RNA Science and Therapeutics

institute: Case Western Reserve University

data_processing:
Illumina Casava1.7 software used for basecalling.
Raw sequencing reads were assessed for quality (FastQC, https://www.bioinformatics.babraham.ac.uk) and de-multiplexed. Low quality reads (PHRED score ≤ 25) were removed. Concatamers and adapter sequences were removed by employing multiple permutations of adapter sequences of varying lengths.
Processed reads were aligned against the mouse genome (mm10) by using bowtie2 using following specifications: Number of mismatches allowed in seed alignment during multi-seed alignment = 1, length of the seed substrings to align during multi-seed alignment = 15, set a function governing the interval between seed substrings to use during multi-seed alignment = S,1,0.50, function governing the maximum number of ambiguous characters (N’s and/or ‘.’s) allowed in a read as a function of read length = L,0,0.15, disallow gaps within this many positions of the beginning or end of the read = 4, set a function governing the minimum alignment score needed for an alignment to be considered `valid` = L, -0.6, -0.6, set the maximum (`MX`) and minimum (`MN`) mismatch penalties, both integers = 6,2, sets penalty for positions where the read, reference, or both, contain an ambiguous character such as `N` = 1, gap opening penalty = 5, gap extension penalty = 3, attempt that many consecutive seed extension attempts to `fail` before Bowtie 2 moves on, using the alignments found so far = 20, set the maximum number of times Bowtie 2 will `re-seed` reads with repetitive seeds = 3. End-to-end alignment mode was used. Only uniquely mapped reads were retained.
Genomic coordinates of the 5’-terminal nucleotide (5’nt) of every mapped read were obtained. Adjacent 5’nt were summed at single nucleotide resolution level by creating a sliding window of 11nt (stride = 1, steps = 5nt on either side or until no new reads were detected), with the 5’nt position at the center.  Crosslinking peaks were defined by plotting the distribution of the count of 5’nt reads in these windows for every location. The peak apex represents the coordinate for the crosslinking peak and the associated coverage value. Error ranges for coverage values corresponding to each crosslinking peak were defined as the 95% confidence interval from the apex of crosslinking peaks.
Coordinates of crosslinking peaks present in all KIN-CLIP libraries, except at the zero timepoint were used to define Dazl binding sites for further analysis. For peaks with coverage at the zero timepoint (~0.2% of peaks), the peak value at t = 0 was subtracted from the KIN-CLIP peaks. Coverage values for each Dazl binding site were converted into a concentration equivalent by normalizing to the amount of bulk crosslinked RNA for each KIN-CLIP library. The normalized read coverage values (in processed data files for each KIN-CLIP condition except 0 sec timepoints as 0 sec values were used for normalization, when applicable) were used for calculating kinetic parameters and other subsequent analyses.
Genome_build: mm10
Supplementary_files_format_and_content: tab delimited text files contain 5' most coordinates (Column 1: Chromosme number Column 2: Coordinate_start Column 3: Coordiante_end) along with normalized coverage values (Column 4). Normalized coverage values were employed for further data analyses and inferences.

source_name: GC-1 spg cells

growth_protocol: GC-1spg cells with inducible DAZL expression were maintained in DMEM high glucose medium (ThermoFisher) supplemented with 10% (v/v) Tet-system approved FBS (Clontech), 100 U/mL penicillin, 100 mg/mL streptomycin, 5 mg/mL blasticidin, and 300 mg/mL Zeocin (all from ThermoFisher) at 37°C, 5% (v/v) CO2.

extract_protocol:
Lysates for generation of cDNA libraries and quantification of Dazl levels were prepared. To quantitate Dazl expression, equal amounts of protein (following lysis) were run on a SDS-PAGE (10% NEXT Gel, Amresco) and transfered to a PVDF membrane. Western blotting was performed with anti-Dazl (Rabbit; 1:5000, US Biological) and anti-Hsp90 (Rabbit;1:10,000; US Biological) antibodies. Chemiluminescence was quantified with the ImagequantTL software.
Cell lysates for each sample were treated with RQ1 DNAse (PromegaM6101) and RNAse A (USB70194Y; at 1: 20,000). Treated Samples werethen subjected to CIP (Roche712023), RNA linker ligation and PNK (NEBM0201S) treatments. The supernatants were loaded onto separate Novex NuPAGE 4-12% Bis-Tris gels, and crosslinked material was transferred to a nitrocellulose membrane. Samples were located on the membrane by autoradiography and RNA-Dazl complexes at 50 - 70 kDa (Dazl molecular weight; 37 kD) were cut. Nitrocellulose fragments were treated with proteinase K (Roche1373196) Dazl bound RNA was isolated, reverse transcribed (SuperScript III (Invitrogen18080051), circularized and amplified to obtain 16 cDNA libraries.

treatment_protocol: Doxycycline induction of Dazl was performed. UV laser crosslinking for defined timepoints and laser power was carried out.

tag: uv laser power

tag: dazl concentration

tag: crosslinking timepoints

tag: molecule subtype

value: Dazl crosslinked RNA


title: A3-9_S24


title: MM3015_24h


title: NIC, E. coli, in vivo (SSII)

organism: Escherichia coli

source_name: Total RNA from E. coli, in vivo treated with NIC

extract_protocol:
Cell pellets were resuspended in 100 µl of Buffer A [10 mM Tris pH 8.0; 100 mM NaCl], supplemented with 5 U SUPERase•In™ RNase Inhibitor, by vigorously vortexing for 30 sec. 25 µl of Buffer B [50 mM EDTA; 120 mM Tris pH 8.0], supplemented with 100 µg/mL final lysozyme, were then added, followed by 30 sec vortexing. Samples were incubated 1 min at room temperature, after which 125 µl of Buffer C [0.5% Tween-20; 0.4% Sodium deoxycholate; 2 M NaCl; 10 mM EDTA] were added to lyse protoplasts. Samples were then incubated at room temperature for 5 min, after which 100 µl of lysate were transferred to 1 mL ice-cold QIAzol and thoroughly mixed by vortexing. 200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min.
Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer [65 mM Tris-HCl pH 8.0; 95 mM KCl; 4 mM MgCl2], then purified with NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 µl (for SSII and HIV RT) or 2.5 µl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 µl 20 µM random hexamers and either 0.25 µl (for SSII) or 0.5 µl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 µl. For HIV RT, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 40 mM MgCl2], 0.5 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT (Worthington Biochemical Corporation, cat. HIVRT). For TGIRT-III, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], 0.25 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT (InGex, cat. TGIRT-50). For SSII, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 0.5 µl DTT 0.1 M, 0.25 µl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III), or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 µg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 µl of a 1:2 dilution of protease inhibitor cocktail (Sigma Aldrich, cat. P9599) in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111L). Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), following manufacturer instructions.

treatment_protocol: A single colony of Escherichia coli K-12 DH10B was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ~ 0.5). 2 mL aliquots were collected by centrifugation at 1000 x g for 5 min. Cell pellets were resuspended in 450 µl 1X PBS (pH 7.4) and 50 µl of each SHAPE compound were added, followed by brief vortexing. Cells were allowed to react at 37°C for 20 min, with moderate shaking, after which 500 µl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 17,000 x g for 2 min (4°C) and supernatant discarded.

tag: strain

value: Escherichia coli K12 DH10B


title: MM3020_24h

tag: tissue


title: 2A3, E. coli, in vivo (SSII)

source_name: Total RNA from E. coli, in vivo treated with 2A3


title: Jag1 wild type_rep1

description: tamoxifen/progesterone

value: stage P0

tag: genotype


title: A2-14_S19


title: A3-2_S21


title: KIN-CLIP Sample 15_1mW laser 1x Dazl 30sec

tag: cell type

value: GC-1spg cells with inducible DAZL expression


title: STR_NT2_7


title: MM3015_4h


title: B5, E. coli, in vivo (SSII)

source_name: Total RNA from E. coli, in vivo treated with B5


title: KIN-CLIP Sample 14_1mW laser 1x Dazl 180sec


title: STR_NT2_3


title: KIN-CLIP Sample 12_1mW laser 4.2x Dazl 0sec


title: 1M4, E. coli, in vivo (SSII)

source_name: Total RNA from E. coli, in vivo treated with 1M4


title: I5, E. coli, in vivo (SSII)

source_name: Total RNA from E. coli, in vivo treated with I5


title: 6A3, E. coli, in vivo (SSII)

source_name: Total RNA from E. coli, in vivo treated with 6A3


title: NAI, E. coli, in vivo (SSII)

source_name: Total RNA from E. coli, in vivo treated with NAI


title: KIN-CLIP Sample 11_1mW laser 4.2x Dazl 30sec


title: A2-16_S20


title: A2-12_S18


title: A2-11_S17


title: A1-11-12_S16


title: MM3016_24h

description:
All time points for the same patient were processed in the same batch.
MM3016_aggr.txt.gz


title: DMSO, E. coli, in vivo (SSII)

source_name: Total RNA from E. coli, in vivo treated with DMSO


title: MM3016_4h


title: 2A3, E. coli, ex vivo (SSII)

source_name: E. coli native deproteinized total RNA, 2A3 treated

extract_protocol:
Samples were then vortexed briefly and 1 mL of ice-cold QIAzol Lysis Reagent was added to each sample, followed by extensive vortexing. 200 µl of chloroform were added, followed by vigorous vortexing for 15 sec. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12,500 x g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 mL tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase at 37°C for 20 min.
Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer [65 mM Tris-HCl pH 8.0; 95 mM KCl; 4 mM MgCl2], then purified with NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, cat. 744970), supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 µl (for SSII and HIV RT) or 2.5 µl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 µl 20 µM random hexamers and either 0.25 µl (for SSII) or 0.5 µl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 µl. For HIV RT, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 40 mM MgCl2], 0.5 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT (Worthington Biochemical Corporation, cat. HIVRT). For TGIRT-III, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl; 15 mM MgCl2], 0.25 µl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT (InGex, cat. TGIRT-50). For SSII, reaction was supplemented with 1 µl 5X RT Buffer [250 mM Tris-HCl pH 8.3; 375 mM KCl], 0.5 µl DTT 0.1 M, 0.25 µl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT (ThermoFisher Scientific, cat. 18064014). Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III), or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 µg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 µl of a 1:2 dilution of protease inhibitor cocktail (Sigma Aldrich, cat. P9599) in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111L). Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, cat. E7645L), following manufacturer instructions.

treatment_protocol: A single colony of Escherichia coli K-12 DH10B was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ~ 0.5). 2 mL aliquots were collected by centrifugation at 1000 x g (4°C) for 5 min. Cell pellets were resuspended in 1 mL of Resuspension Buffer [15 mM Tris-HCl pH 8.0; 450 mM Sucrose; 8 mM EDTA], and lysozyme was added to a final concentration of 100 µg/mL. After incubation at 22°C for 5 min and on ice for 10 min, protoplasts were collected by centrifugation at 5000 x g (4°C) for 5 min. Pellets were resuspended in 120 µl Protoplast Lysis Buffer [50 mM HEPES pH 8.0; 200 mM NaCl; 5 mM MgCl2; 1.5% SDS], supplemented with 0.2 µg/µl Proteinase K. Samples were incubated for 5 min at 22°C and for 5 min on ice. Sample was then extracted 2 times with phenol:chloroform:isoamyl alcohol (25:24:1, pre-equilibrated 3 times with RNA folding buffer [50 mM HEPES pH 8.0; 200 mM NaCl; 5 mM MgCl2]), and once with chloroform. 20 U SUPERase•In™ RNase Inhibitor were then added and RNA was equilibrated at 37°C for 20 min prior to probing. 180 µl of deproteinized E. coli rRNAs in RNA folding buffer were mixed with 20 µl of each SHAPE compound (assuming complete conversion, stocks should be ~1 M, resulting in a final concentration of 100 mM in the probing reaction). RNA was then allowed to react at 37°C for 15 minutes, with moderate shaking, after which 200 µl of 1 M DTT were added, to quench the reaction.


title: KIN-CLIP Sample 10_1mW laser 4.2x Dazl 180sec


title: A1-13_S15


title: KIN-CLIP Sample 9_1mW laser 4.2x Dazl 680sec


title: KIN-CLIP Sample 13_1mW laser 1x Dazl 680sec


title: NIC, E. coli, ex vivo (SSII)

source_name: E. coli native deproteinized total RNA, NIC treated


title: MM3012_Ctrl

description:
All time points for the same patient were processed in the same batch.
MM3012_aggr.txt.gz

source_name: PBMC

value: Ctrl


title: A1-12_S14

value: Male


title: MM3016_Ctrl


title: KIN-CLIP Sample 8_2.6mW laser 1x Dazl 0sec


title: MM3016_72h


title: MM3012_72h


title: A1-10_S13

value: Plasma


title: B5, E. coli, ex vivo (SSII)

source_name: E. coli native deproteinized total RNA, B5 treated


title: MM3012_24h


title: KIN-CLIP Sample 7_2.6mW laser 1x Dazl 30sec


title: 6A3, E. coli, ex vivo (SSII)

source_name: E. coli native deproteinized total RNA, 6A3 treated


title: MM3012_4h


title: 1M4, E. coli, ex vivo (SSII)

source_name: E. coli native deproteinized total RNA, 1M4 treated


title: NAI, E. coli, ex vivo (SSII)

source_name: E. coli native deproteinized total RNA, NAI treated


title: A1-6_S11

source_name: plasma sample

value: Kiss treated


title: I5, E. coli, ex vivo (SSII)

source_name: E. coli native deproteinized total RNA, I5 treated


title: A1-7_S12


title: MM3009_Ctrl

description:
All time points for the same patient were processed in the same batch.
MM3009_aggr.txt.gz


title: A2-10_S10

value: Female


title: A2-9_S9


title: DMSO, E. coli, ex vivo (SSII)

source_name: E. coli native deproteinized total RNA, DMSO treated


title: MM3009_24h


title: KIN-CLIP Sample 3_2.6mW laser 4.2x Dazl 30sec

source_name: GC-1 spg cells


title: KIN-CLIP Sample 5_2.6mW laser 1x Dazl 680sec


title: A2-7_S8


title: KIN-CLIP Sample 4_2.6mW laser 4.2x Dazl 0sec


title: NAI, E. coli, ex vivo (HIV RT)


title: MM3009_4h


title: A2-5_S7


title: KIN-CLIP Sample 1_2.6mW laser 4.2x Dazl 680sec


title: MM3008_Ctrl

description:
All time points for the same patient were processed in the same batch.
MM3008_aggr.txt.gz


title: DMSO, E. coli, ex vivo (HIV RT)


title: KIN-CLIP Sample 6_2.6mW laser 1x Dazl 180sec


title: A2-2_S6


title: NAI, E. coli, ex vivo (TGIRT-III)


title: MM3009_72h


title: A1-11-9_S5


title: MM3008_72h


title: DMSO, E. coli, ex vivo (TGIRT-III)


title: MM3008_24h


title: A1-4_S4


title: MM3005_Ctrl

description:
All time points for the same patient were processed in the same batch.
MM3005_aggr.txt.gz


title: MM3008_4h


title: A1-1_S1


title: A1-2_S2


title: Treated(3)

city: Oxford

first: Xiaodong

last: Zhuang

state: Oxfordshire

address: Old Road Campus, Roosevelt Drive

country: United Kingdom

institute: University of Oxford

description:
Treat3
FRG-NOD mice transplanted with primary human hepatocytes.

data_processing:
Sequence adapters were removed and reads were quality trimmed by Trim Galore v0.5.0.
The reads were mapped against the reference mouse genome (mm10/GRCm38) and reference human genome (hg38/GRCm38) using STAR v2.5.3.
Counts per gene were calculated using Rsubread v1.28.1.
Reads were analysed by edgeR v3.30.0, first normalised using TMM and then counts per million were calculated and differential expression analysis was performed. Mouse genes were converted to human orthologs using biomaRt v2.44.0 and the built-in Ensembl datasets for human and mouse.
Genome_build: mm10/GRCm38 (mouse); hg38/GRCm38 (human)
Supplementary_files_format_and_content: genes.readcount.annot.txt: Tab-delimited text file includes read counts for each Sample.
Supplementary_files_format_and_content: genes.FPKM.annot.txt: Tab-delimited text file includes FPKM values for each Sample.
Supplementary_files_format_and_content: *_DE.csv: Comma-separated text files include differential expression analysis.

source_name: Liver

extract_protocol:
Livers were removed, flash frozen on dry ice, and RNA was extracted using Qiagen RNeasy kit. 2 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: FRG-NOD

tag: infection

value: 10^9 HBV genome equivalents purified from supernatant of HepG2.2.15 cell line

value: Liver


title: Treated(1)

description:
Treat1
FRG-NOD mice transplanted with primary human hepatocytes.


title: MM3005_4h


title: Control(3)

description:
Ctrl3
FRG-NOD mice transplanted with primary human hepatocytes.

value: Vehicle


title: MM3001_Ctrl

description:
All time points for the same patient were processed in the same batch.
MM3001_aggr.txt.gz


title: Control(2)

description:
Ctrl2
FRG-NOD mice transplanted with primary human hepatocytes.


title: Control(1)

description:
Ctrl1
FRG-NOD mice transplanted with primary human hepatocytes.


title: v16a_h3k27ac

city: Toronto

first: Musaddeque

last: Ahmed

state: ON

address: 101 College St

country: Canada

institute: University health Network

data_processing:
The raw sequencing data were mapped to human genome assembly Hg19 using BowTie2
The peaks were called using MACS2
Genome_build: Hg19
Supplementary_files_format_and_content: Peaks called by MACS2

growth_protocol: Cells were grown in 10% FBS

extract_protocol:
Genomic DNA was extracted
standard protocol

tag: cell line background

tag: source tissue

value: Prostate

tag: chip anitbody


title: sgMYCpromoter-2-_V16

data_processing:
The raw sequencing data were mapped to human genome assembly Hg19 using TopHat2 version 2.1.0 (Kim et al., 2013) in its default setting.
The reads per gene were counted using HTSeq version 0.7.2 (Anders et al., 2015) against refGene gene annotation
The differential expression analyses were performed using DESeq2 package in R
Genome_build: Hg19
Supplementary_files_format_and_content: Raw count matrix of refGene genes in all samples

source_name: sgMYC promoter_V16A

growth_protocol: 24hrs post-transduction, cells were selected with completed medium containing puromycin for 72hrs.

extract_protocol:
Total RNA was extracted using the RNeasy Mini Kit (74106, QIAGEN)
Following on-column DNase digestion RNA-seq libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (RS-122-2101, Illumina)

treatment_protocol: 22Rv1 and V16A dCas9-KRAB stable cells were transduced individually with lentiviral particles containing two sgRNAs against the MYC enhancer, MYC promoter or a non-target region (Luciferase and LacZ).

tag: genotype/variation

value: sgRNAs against the MYC promoter


title: MM3001_72h

value: PBMC

tag: group


title: sgMYC_Promoter_2_-_22RV1

description: sgMYC_Promoter_2_-_22RV1_CRISPRi_S6_L001


title: Treated(2)

description:
Treat2
FRG-NOD mice transplanted with primary human hepatocytes.

tag: treatment


title: heart-allogeneic heart graft-rep3

city: Harbin

first: Maomao

last: Zhang

address: No.246  Xuefu Road, Nangang District

country: China

institute: Second Affiliated Hospital of Harbin Medical University

hyb_protocol: 1 μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, then heated at 60°C for 30 min, and finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100 μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.

scan_protocol: The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).

data_processing: Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, genes that at least 1 out of 6 samples have flags in Detected (“All Targets Value”) were chosen for further data analysis. Differentially expressed genes with statistical significance between the two groups were identified through Volcano Plot filtering. Differentially expressed genes between the two samples were identified through Fold Change filtering. Hierarchical Clustering was performed using the R scripts. GO analysis and Pathway analysis were performed in the standard enrichment computation method.

source_name: heart tissue,allogeneic heart graft,5day,replicate 3

label_protocol: Total RNA from each sample was linearly amplified and labeled with Cy3-UTP. The Labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.

growth_protocol: Eight to ten-week-old male C57BL/6J and BALB/c mice were obtained from the animal experimental center of Harbin Medical University. All animal procedures were approved by the Animal Care Ethics Committee of the Second Affiliated Hospital, Harbin Medical University, and were performed in line with the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23 revised 2011, USA).

extract_protocol: The heart underwent collagenase digestion followed by filtration through 40μm filters to remove extracellular connective tissue debris before centrifugation and reconstitution. RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis.

treatment_protocol: Syngeneic (C57BL/6→C57BL/6) and allogeneic (BALB/c→C57BL/6) heterotopic heart transplants were conducted. Transplanted heart grafts were harvested on day 5 post-transplantation.

value: heart

tag: gender

value: male


title: hESC_lung_2

first: Joyce

middle: huanhuan

last: Chen

state: IL

address: 5801 S Ellis Ave, Chicago

department: Pritzker School of Molecular Engineering

institute: Uchicago

data_processing:
The sequencing data were primarily analyzed by the 10X cellranger pipeline (v3.0.2)
Cellranger mkfastq was used for sample demultiplexing and fastq generation.
Cellranger count was used to align reads to either the 10X pre-built human reference genome (GRCh38 v3.0.0) for hESC_lung_1&2.
Genome_build: 10X pre-built human reference genome (GRCh38 v3.0.0) for lung cell_1 and 2.
Supplementary_files_format_and_content: matrix.mtx.gz stores UMI counts per gene per cell; features.tsv.gz stores gene annotations; barcodes.tsv.gz stores cell barcodes.

source_name: Lung cells

growth_protocol: The hESC line-RUES2 was cultured on irradiated mouse embryonic fibroblasts (Global Stem) at a density of 20,000- 25,000 cells/cm2 in a medium of DMEM/F12, 20% knockout serum replacement (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma Aldrich) and 20 ng/ml bFGF (R&D Systems), and medium was changed daily. hESC cultures were maintained in an undifferentiated state at 37 °C in a 5% CO2/air environment until stem cells reached about 90% confluence.

extract_protocol:
Single-cell suspensions were generated by dissociating the cultured RUES2 cells with 0.05% Trypsin/0.02% EDTA for 10-15 min, followed with passing through 40µM strainer.
Single-cell capture, reverse transcription, cell lysis, and library preparation was performed using the Single Cell 3' version 2 kit and chip according to the manufacturer’s protocol (10x Genomics)

treatment_protocol: RUES2 cells were differentiated to endoderm, anterior foregut endoderm, lung progenitor cells and functional lung cells using growth factor cocktails inclduing Activin A, CHIR, DSW, IWP2, FGF10, KFG, DAPT, etc. At day 25, a portion of cells from late stage lung progenitor cells was harvested for examination. The other cultures were allowed to continue to mature lung cells  (day 25 to 55) in maturation media for induction of the major types of cells found in human  lung epithelium.

tag: cell line

value: RUES2 human embryonic stem cell

value: RUES2-derived lung cells

tag: time


title: heart-allogeneic heart graft-rep1

description: Transplanted heart grafts were harvested on day 5 post-transplantation.

source_name: heart tissue,allogeneic heart graft,5day,replicate 1


title: heart-syngeneic heart graft-rep2

source_name: heart tissue,syngeneic heart graft,5day,replicate 2


title: heart-allogeneic heart graft-rep2

source_name: heart tissue,allogeneic heart graft,5day,replicate 2


title: heart-syngeneic heart graft-rep1

source_name: heart tissue,syngeneic heart graft,5day,replicate 1


title: sgMYCpromoter-1-_V16A


title: hESC_lung_1


title: heart-syngeneic heart graft-rep3

source_name: heart tissue,syngeneic heart graft,5day,replicate 3


title: MM3001_24h

source_name: PBMC


title: 201B7_astroP1

city: Tokyo

first: Nicolas

last: Leventoux

state: Tokyo

address: 35, Shinanomachi

department: Physiology

institute: Keio University

data_processing:
Libraries were sequenced using a NovaSeq6000 (Illumina) to obtain 150-bp paired-end reads.
Raw FASTQ files were trimmed for adapters by Cutadapt and aligned to the GRCh37 genome build using HISAT2.
Counts were calculated using featureCounts, and normalized by vst transformation using the DESeq2.
Genome_build: GRCh37
Supplementary_files_format_and_content: tab-delimited text files include count values for each Sample

source_name: neurospheres Passage1

growth_protocol: iPSC grew in DMEM/F12 + KSR + NEAA + 2-mercaptoethanol and hFGF ; EB grew in DMEM/F12 + KSR + NEAA + 2-mercaptoethanol and L-Gluta + DM, SB and CHIR then with RA then with PM ; Spheres grew in KBM + NEAA + B27 + FGF + EGF and PM ; iPast grew in MHM + B27 + NEAA + BDNF and GDNF

extract_protocol:
We extracted RNA using the RNeasy kit (QIAGEN) according to manufacturer's protocol.
The indexed cDNA libraries were prepared using the Nextera XT library preparation kit (Illumina)

treatment_protocol: iPSC were dissociated by trypsin and collagenase ; spheres and iPast by TrypLE Select.

value: iPSC-derived neurospheres passage 1


title: 201B7_astroP2

source_name: neurospheres Passage2

value: iPSC-derived neurospheres passage 2


title: KIN-CLIP Sample 2_2.6mW laser 4.2x Dazl 180sec

value: GC-1spg cells with inducible DAZL expression

tag: uv laser power

tag: dazl concentration

value: Dazl crosslinked RNA


title: NC rep2

city: Hefei

first: xiaoyuan

last: song

address: Huangshan Road

institute: University of Science & Technology of China

data_processing:
Reads were mapped using HISAT2 (version 2.1.0) to the GRCM38 Ensembl genome.
Sort and convert the SAM files to BAM files by using SAMtools (version 1.7).
The mapped reads were then counted using featureCounts (version 2.0.1) with Mus_musculus.GRCm38.101.gtf annotations
Differentially expressed genes were determined using R package DESeq2 (version 1.26.0)
Genome_build: GRCM38
Supplementary_files_format_and_content: tab-delimited text files include gene counts for every gene and every sample
Supplementary_files_format_and_content: tab-delimited text files include differentially expressed genes produced by DESeq2

source_name: HT22 cells

growth_protocol: HT22 were routinely cultured in DMEM medium with 10% fetal bovine serum, Antibiotic-Antimycotic, and incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The cells were subcultured every 2 days.

extract_protocol:
Total RNA were extracted using TRIzol Reagent
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample

treatment_protocol: Cells were seeded in 12‐well culture plates and transfected with siRNA when cells had reached 30% to 40% confluences. Then, cells were transfected with 10 nΜ GAS5 siRNA or NC siRNA using INTERFERin reagent (Polyplus, 409-10, France) at a 1 of 250 final dilution for 48 hours.

value: Hippocampal neuronal

value: untreated


title: MM3005_24h


title: 201B7_EB

source_name: embryoid bodies

value: iPSC-derived embryoid bodies


title: sgMYC_Promoter_1_-_22RV1

description: sgMYC_Promoter_1_-_22RV1_CRISPRi_S5_L001


title: MM3001_4h


title: sgMYCenhancer_2_-_22RV1

value: sgRNAs against the MYC enhancer


title: KD1

value: treated with GAS-siRNA210


title: H58SACT

city: Chapel Hill

first: Kenichi

last: Okuda

state: NC

address: 125 Mason Farm Road

department: Department of Medicine, Cystic Fibrosis/Pulmonary Research and Treatment Center

institute: University of North Carolina at Chapel Hill

data_processing:
Illumina bcl2fastq v2.19 used for basecalling.
Sequence reads in FASTQ files were mapped to GRCh38 reference genome with GENCODE 32 gene annotation by HISAT2, and gene expression estimates were derived from StringTie using default parameters.
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample.

source_name: Small airway epithelial cells

growth_protocol: Matched large airway epithelial (LAE) and small airway epithelial (SAE) cells were isolated from freshly excised normal human lungs from transplant donors with lungs unsuitable for transplant. Isolated LAE and SAE cells were separately expanded using a modified conditional reprogramming cell method, and then grown to obtain well-differentiated air-liquid interface (ALI) cultures

extract_protocol:
For RNA extraction from cultured LAE and SAE cells, TRI Reagent was used according to the manufacturer’s instructions. RNA concentrations were determined using a NanoDrop-8000 spectrophotometer. Total RNA was used for bulk RNA-seq.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: After ALI 28 days, LAE and SAE cells were utilized for RNA isolation for RNAseq.

tag: donor code

value: Primary small airway epithelial cells

value: Primary cells

tag: age

value: ALI day 28

value: No treatment

tag: disease state

value: Normal


title: JS181116

city: West Haven

first: Sarah

last: Slavoff

state: CT

address: 300 Heffernan Dr

department: Chemistry

institute: Yale University

description: Reads filtered for number of T-to-C mutations (0-5+) and strandedness (min/pos)

data_processing:
Reads were filtered for unique sequences using FastUniq
Reads were trimmed using cutadapt (--minimum-length=20)
Reads were aligned to the GRCh38 genome using STAR aligned with default parameters and --mp 4,2 .
Reads were processed using Picard tools, including FixMateInformation, SortSam and BuildBamIndex
Reads were filtered for those that aligned uniquely (flag: 83/163, 99/147), with MAPQ ≥ 2, and without insertions.
HTSeq-count was used to identify mapped reads in UCSC transcripts (union mode)
T-to-C mutations were identified using Rsamtools and a custom R script (available upon request). Only mutations at positions with a base quality score of greater than 45 that were at least three nt from the end of the read were counted. Reads were excluded where there were greater than five T-to-C mutations and these mutations did not account for at least one third of the observed mutations (NM tag).
SNP sites were filtered from our data in two ways: 1, bcftools was used to identify T-to-C SNP sites in control samples, and these sites were excluded from analysis; 2, sites exhibiting high T-to-C mutation rates in non-s4U treated controls were excluded from analysis.
To examine the distribution of reads with each minimum number of T-to-C mutations, bam files were filtered using Picard tools.
STAR aligner was used to make genome-coverage tracks (inputAlignmentsFromBam mode, outWigType bedGraph), and tracks were normalized using DESeq2 (estimateSizeFactors).
Tracks were converted to binary format using IGVtools (toTDF).
Genome_build: GRCh38
Supplementary_files_format_and_content: bedGraph track
Supplementary_files_format_and_content: Name of the TDF files reflect the number of T to C mutations identified.
Supplementary_files_format_and_content: TC.0 = zero
Supplementary_files_format_and_content: TC.1 = 1
Supplementary_files_format_and_content: TC.2 = 2
Supplementary_files_format_and_content: etc

source_name: immortalized 293Ts

growth_protocol: 293T cells were grown in DMEM containing 1% penicillin/streptomycin, 10% FBS, at 37°C.

extract_protocol:
RNA was harvested with Trizol reagent, followed by chloroform extraction and ethanol precipitation. Genomic DNA was depleted using Turbo DNAse, followed by cleanup with RNAclean beads. RNA was treated with oxidative-nucleophilic-aromatic-substitution chemistry as described in Schofield et al. Nature Methods 2018.
Libraries were generated following standard protocols using the SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian, V2 (Takara Bio USA, cat. 634413).

treatment_protocol: 293T cells were pre-treated with indicated compounds at 2 μM for 5 h. s4U plus and minus cells had their media replaced with growth media supplemented with 500 uM s4U or left unsupplemented, respectively. Cells were incubated with s4U for 2h.

tag: strain

value: DCP2 KO

tag: s4u feed

value: No


title: sgMYCenhancer_2-_V16A

value: Prostate


title: NC rep1

tag: tissue


title: Cisplatin rep2

first: Lindsey

last: Seldin

address: 465 21st Avenue South U3200 MRBIII

data_processing:
base-calling, alignment, filtering, peak-calling, and generation of normalized abundance measurements were all performed by CLC Genomics Workbench 12.0.2 software
Genome_build: Mus_musculus.GRCm38.97
Supplementary_files_format_and_content: each *paired* processd data file contains two spreadsheets, the "GE" tab (left one) with gene expression information and the "TE" tab (right one) with transcript expression information, and it contains the RPKMs, TPMs and gene reads for each sample. The 'differential expression*' file contains the fold change values between conditions and p-values

source_name: Cisplatin_dermal fibroblasts

extract_protocol:
Fibroblasts were isolated from C3H wild-type mouse backskin 4 d following vehicle and Cisplatin intradermal injections. RNA isolation was performed using the RNeasy Mini Kit.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Adult

value: Cisplatin intradermal injection

value: Backskin dermis

tag: cell type

value: Dermal fibroblasts


title: JS181115

value: Wild Type


title: JS181112

value: Yes


title: sgMYCenhancer_1-_V16A

tag: cell line background

tag: source tissue


title: sgMYCenhancer_1_-_22RV1

tag: genotype/variation


title: KD2 rep2

value: Hippocampal neuronal

value: treated with GAS-siRNA596


title: H47SACT


title: sgLuc-_V16A [sgCtr2]

value: sgRNAs against non-target region


title: sgLuc_-_22RV1 [sgCtr2]


title: JS181111


title: Vehicle rep1

source_name: vehicle_dermal fibroblasts

value: vehicle


title: H45SACT

value: Primary small airway epithelial cells

value: Primary cells


title: H42SACT

value: ALI day 28

value: No treatment


title: Vehicle rep2


title: JS181109

value: DCP2 KO


title: KD2 rep1


title: H41SACT

value: Normal


title: JS181110


title: JS181106


title: sgLacZ_-_22RV1 [sgCtr1]

value: Prostate


title: Salmonella Derby ER278ΔhilD - rep3

city: Parma

first: Stefano

last: Pongolini

state: PR

address: Strada dei Mercati 13/A

department: Risk Analysis and Genomic Epidemiology Unit

country: Italy

institute: Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna

data_processing:
Trim Galore! was used for quality and adapter trimming as well as quality control.
Quality checked transcriptomic reads from each sample were mapped to ER1175 draft genome (ERS3898662) by HISAT-2.0.4.
HTseq was used to estimate the number of reads for each sample mapped on each annotated gene.
Detection of differentially expressed genes was made using the package DESeq2.
Genome_build: ERS3898662 (at ENA)
Supplementary_files_format_and_content: tab-delimited text file include normalized number of reads per CDS for each Sample

organism: Salmonella enterica subsp. enterica serovar Derby

source_name: Total RNA from bacteria

growth_protocol: Strains were grown overnight in 5 mL LB broth and diluted to OD600 = 0.1 in 250 mL flasks in 20 mL LB broth. Flasks were incubated at 37°C and 220 rpm until OD600 = 2.0.

extract_protocol:
0.6 mL of each culture (about 1.2 x 109 CFU/mL) was centrifuged at 16,000g for 2 minutes and pellets were frozen in a dry ice ethanol bath. Cells, resuspended in Lysis Buffer, were processed in a FastPrep-24 Instrument for 90 seconds, at 6.0 m/s, for cell disruption. RNA extraction was performed by FastRNA SPIN Kit for Microbes (MP Biomedicals) according to the manufacturer instruction. Trace DNA was removed by DNase digestion with TURBO DNA-free kit (Invitrogen Ambion), using rigorous treatment. Complete loss of genomic DNA after DNase treatment was verified by PCR on RNA samples using rpoD_FW and rpoD_RV primers. RNA samples were then precipitated for purification. RNA quality was assessed using a 2100 Bioanalyzer (Agilent) according to Agilent RNA 6000 Nano Kit protocol. Each sample was then retro-transcribed by SuperScript VILO cDNA Synthesis Kit (Invitrogen). In order to obtain double-strand cDNA for libraries preparation, single-strand cDNA samples were purified using 1.8X Agencourt AMPure XP Beads (Beckman Coulter) and the second cDNA strand was synthesized using NEBNext Ultra II Non-Directional RNA Second Strand Synthesis. A second step of beads-based purification was performed before libraries preparation.
Libraries were prepared using Nextera XT V2 kit according to the reference guide.

tag: genotype

value: delta_hilD_a::cat; CmR


title: JS181105

value: Wild type

tag: treatment


title: H39SACT


title: Salmonella Derby ER278ΔhilD - rep2


title: H38SACT


title: a549_day16_rep2

description: guide RNA

data_processing:
The fastq files were first converted to fasta file using custom shell script. For each sample, a custom bowtie database was generated by the command bowtie-build in bowtie suite (version 1.1.2). The library sgRNAs were mapped against the database for each sample using bowtie with the parameter v=0 and default values for other parameters.
Each targeted region in the library was split into 100 bp sliding windows (50 bp offset) ensuring at least two sgRNAs target a window. The differential sgRNA abundance was estimated using the tool MAGeCK (Li et al., 2014). The 100 bp window with the lowest p-value in each CRE is treated as a representative of the essentiality of the CRE. For adjacent 400 bp windows, we merge all windows into the most essential window. The depletion score of each CRE is the “neg.score” as reported by MAGeCK.
Genome_build: Hg19
Supplementary_files_format_and_content: MAGeCK output for all cell lines.  The first column is the library region, the next nine columns indicate the p value, log2 fold change and depletion score of differential analyses between day 16 and 0 in V16A, 22Rv1 and A549 cells, respectively. The last column indicate the class of the region (CRE or control regions).

growth_protocol: A549, 22Rv1 and V16A cells were cultured in RPMI1640 medium with 10% FBS (Wisent) and 1% Penicillin and Streptomycin (450-201-EL, Wisent). All cells were cultured at 37 degrees in 5% CO2. All cell lines were authenticated by STR and routinely tested for mycoplasma using the EZ-PCR mycoplasma Test Kit.

extract_protocol: Samples were collected in replicates on Day 0 and Day 16 post- puromycin selection for genomic DNA analysis.

treatment_protocol: To introduce expressiond of dCas-KRAB, each cell line was titrated with library virus generated from hU6-sgRNA hUbC-dCas9-KRAB-T2a-Pur (Addgene plasmid # 71236). MOI was determined by infecting ~5-6 million cells with varying amounts of library virus for 24 hours, which were then split into media with or without puromycin for 48-72 hours (A549, 3.5 ug/mL; 22Rv1, V16A, 3 ug/mL; LNCaP, 2.5 ug/mL). A ratio between these two populations was calculated to determine the infection efficiency to achieve a MOI of ~0.3. The amount of library virus was scaled up along with the number of cells to ensure that on average every sgRNA was represented in ~300 cells. For each screen, cells were split into triplicates every 3-4 days, and maintained at 300x coverage throughout the screen.

value: Lung

value: dCas9-KRAB expressing

tag: time point


title: JS181104

value: Wild Type


title: Cisplatin rep1

institute: Vanderbilt University

value: Adult

value: Cisplatin intradermal injection

value: Backskin dermis

value: Dermal fibroblasts


title: Salmonella Derby ER278ΔhilD - rep1


title: H32SACT


title: Salmonella Derby ER1175ΔhilD - rep3

value: delta_hilD_v::cat; CmR


title: CRISPR library

city: Chicago

first: Allen

last: Zhu

address: 929 E 57th St, Room E331

institute: University of Chicago

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were first mapped to the mycoplasma genome to check for contamination and remove any reads (parameter -k 7), then mapped to the mm10 whole genome using Hisat2 v2.1.0 with parameters -k 1
For m6A-seq, our lab's software packages RADAR (https://scottzijiezhang.github.io/RADARmanual/Mannual.html) and MeRIPtools (https://scottzijiezhang.github.io/MeRIPtoolsManual/index.html) were used to call peaks. QNB was used to perform differential methylation analysis.
For RNA-seq, we used the input samples only. DESeq2 was used to count reads for each gene and to perform differential expression analysis.
Genome_build: mm10
Supplementary_files_format_and_content: DEG-all.csv: DESeq2
Supplementary_files_format_and_content: allPeakResults.csv: MeRIPtools
Supplementary_files_format_and_content: m6A-diff-Me-Peaks.txt: QNB

source_name: CRISPR library

extract_protocol:
Cells and RNA was harvested using Trizol reagent. Total RNA was extracted and underwent one round of poly-A Dynabeads purification (ThermoFisher). RNA was then fragmented, and Input saved. Fragmented RNA then underwent m6A-immunoprecipitation. Illumina TruSeq v2 RNA Sample Prep Kit (Cat#20020595) was used for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols
m6A MeRIP-seq

tag: sample type

value: CRISPR library


title: H10SACT

value: Primary small airway epithelial cells

value: Primary cells


title: JS181103

value: Yes


title: a549_day16_rep1

description: guide RNA


title: a549_day0_rep2

description: guide RNA


title: HeLa Low-GFP rep 1

source_name: HeLa cells

value: HeLa

value: cervical cancer


title: H58LACT

source_name: Large airway epithelial cells

value: Primary large airway epithelial cells

value: ALI day 28

value: No treatment

value: Normal


title: Salmonella Derby ER1175ΔhilD - rep2


title: Salmonella Derby ER1175ΔhilD - rep1


title: H09SACT


title: 22rv1_day16_rep2

description: guide RNA

value: Prostate

value: dCas9-KRAB expressing


title: Salmonella Derby ER278::hilD_v - rep3

value: delta_hilD_a::hilD_v-cat; CmR


title: HeLa High-GFP rep 1


title: Salmonella Derby ER278::hilD_v - rep2


title: 22rv1_day16_rep1

description: guide RNA


title: N5_control

city: Shanghai

first: Zhou

last: haiwen

address: 500 Qu-xi Road, Shanghai

department: Oral Mucosal Diseases

institute: Shanghai Ninth People’s Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine

description:
processed_data_control_samples.xlsx
GSM3765316

data_processing:
Basecalls performed using CASAVA version 1.8
circRNA-seq reads were mapped to human genome by BWA-MEM software by default parameters except -T = 19
circRNAs were detected and annotated with CIRI software
High throughput sequencing service was provided by CloudSeq Biotech (Shanghai, China). Transcriptome high throughput sequencing and subsequent bioinformatics analysis were all done by Cloud-Seq Biotech (Shanghai, China). Briefly, total RNA was used for removing the rRNAs using Ribo-Zero rRNA Removal Kits (Illumina, USA) following the manufacturer's instructions. RNA libraries were constructed by using rRNA-depleted RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) according to the manufacturer’s instructions. Libraries were controlled for quality and quantified using  the BioAnalyzer 2100 system (Agilent Technologies, USA).Libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 150 cycles on Illumina HiSeq Sequencer according to the manufacturer’s instructions.
Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3). The high quality trimmed reads were used to analyse circRNAs. The high quality reads were aligned to the reference genome/transcriptome with STAR software (v2.5.1b) and circRNAs were detected and identified with DCC software (v0.4.4). edgeR software (v3.16.5) was used to normalized the data and perform differentially expressed circRNA analysis.
Genome_build: hg19
Supplementary_files_format_and_content: excel format: Number of raw junction read

source_name: healthy oral mucosa tissue

growth_protocol: Patients with previous histories of All tissue samples were immediately frozen in liquid nitrogen after resection and stored at -80℃ until RNA retraction

extract_protocol:
RNA extraction protocol
library construction protocol

tag: diagnosis

value: healthy control

tag: time

value: fresh

value: oral mucosa


title: H47LACT


title: H45LACT


title: sgLac_-_V16A [sgCtr1]

value: sgRNAs against non-target region


title: N4_control

description:
processed_data_control_samples.xlsx
GSM3765315


title: 22rv1_day0_rep2

description: guide RNA


title: Salmonella Derby ER278::hilD_v - rep1


title: H42LACT


title: 22rv1_day0_rep1

description: guide RNA


title: Salmonella Derby ER1175::hilD_a - rep3

value: delta_hilD_v::hilD_a-cat; CmR


title: N3_control

description:
processed_data_control_samples.xlsx
GSM3765314


title: H41LACT


title: a549_day0_rep1

description: guide RNA

value: Lung


title: v16a_day16_rep2

description: guide RNA


title: H39LACT

value: Primary cells


title: N1_control

description:
processed_data_control_samples.xlsx
GSM3765312


title: v16a_day16_rep1

description: guide RNA


title: H38LACT


title: Salmonella Derby ER1175::hilD_a - rep2


title: v16a_day0_rep2

description: guide RNA


title: Salmonella Derby ER1175::hilD_a - rep1


title: H32LACT


title: K6

description: CircRNA Expression Profiling

source_name: oral leukoplakia tissue

value: oral leukoplakia


title: Salmonella Derby ER278 - rep3

value: wild type


title: v16a_day0_rep1

description: guide RNA


title: H10LACT


title: K5

description: CircRNA Expression Profiling


title: Salmonella Derby ER278 - rep1


title: K4

description: CircRNA Expression Profiling


title: K3

description: CircRNA Expression Profiling


title: WA09-C3 (day 80)_r2

city: Fort Worth

first: Yu-Chieh

last: Wang

state: Texas

address: 3500 Camp Bowie Boulevard

department: Department of Pharmaceutical Sciences

institute: University of North Texas Health Science Center

data_processing:
Cell Ranger pipeline (version 2.0.1, 10x Genomics) was performed to enable alignment, de-duplication, and filtering, feature-barcode matrices of all samples, and a single feature-barcode matrix (an aggregation) containing all the data
Output of the Cell Ranger pipeline was taken into Seurat for further QC and cell type determination and differential gene expression analysis.
Genome_build: grch38.91
Supplementary_files_format_and_content: A MatrixMarket format text file that includes UMI counts for all samples and the relevant tab-delimited files

source_name: cerebral organoids

growth_protocol: Cerebral organoids were generated using WA09 hESC subclones (with and without the edited NGLY1 gene) and NGLY1-deficient patient's hiPSCs (with and without the ectopic expression of human NGLY1.  The detailed protocol for culturing cerebral organoids in the neural induction, neural differnetiation and cortical expansion media is described in the relevant pulication.

extract_protocol: RNA libraries were prepared for sequencing using Chromium Single Cell 3' Reagent (v2) Kits, Chromium Single Cell 3' Library & Gel Bead Kits, Chromium Single Cell A Chip Kit, and Chromium i7 Multiplex Kits from 10x Genomics according to the manufacturer's protocol.

tag: cell types

value: neural cells developed in cerebral organoids

tag: developmental stages

value: 80 days

tag: ngly1 condition

value: NGLY1-deficient (NGLY1 gene edited)


title: WA09-C6 (day 80)

value: NGLY1-functional (gene-editing control)


title: Salmonella Derby ER1175 - rep3


title: WA09-C3 (day 80)_r1


title: N2_control

description:
processed_data_control_samples.xlsx
GSM3765313


title: Salmonella Derby ER1175 - rep2


title: K2

description: CircRNA Expression Profiling


title: dd49_dep

city: Los Angeles

first: Gianni

last: Carraro

state: CA

address: 8700 Beverly Blvd

department: Medicine

institute: Cedars-Sinai Medical Center

description: 10X Genomics Single Cell RNA Sequencing

data_processing:
Raw reads were aligned to the transcriptome by Cellranger. Expression counts for each gene in all samples were collapsed and normalized to unique molecular identifier (UMI) counts.
Genome_build: GRCh38
Supplementary_files_format_and_content: Processed data for each sample are provided in binary format HDF5 as produced by Cellranger
Supplementary_files_format_and_content: R data set object: This object contains also datasets of proximal airway from GSE143706 (GSM4272915 - 920)

source_name: Airway Epithelium  distal

extract_protocol:
Tissue was minced, digested with Liberase, and epithelial cells were fractionated by FACS  (DAPI-,CD45-,CD31-,CD326+).
The barcoded sequencing libraries were quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). Sequencing libraries were loaded on a NovaSeq 6000 (Illumina).

value: Cells isolated from dissected and FACS sorted human airway material

tag: disease

value: Normal


title: Salmonella Derby ER1175 - rep1

tag: strain


title: K1

description: CircRNA Expression Profiling


title: dd10_dep

description: 10X Genomics Single Cell RNA Sequencing


title: dd39_dep

description: 10X Genomics Single Cell RNA Sequencing


title: Salmonella Derby ER278 - rep2


title: WA09-C6 (day 40)

value: 40 days


title: LN6

city: Shenyang

first: hua

last: Zhou

address: 155 North Najing St, Shenyang, Liaoning, China

institute: The First Hospital of China Medical University

description: circular RNA

data_processing:
CircRNA-Seq high throughput sequencing and subsequent bioinformatics analysis were all done by Cloud-Seq Biotech (Shanghai, China). Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30.
After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3). The reads were aligned to the reference genome/transcriptome with STAR software and circRNAs were detected and annotated with DCC software
circBase database and circ2Trait disease database were used to annotated the identified circRNAs. Raw junction reads for all samples were normalized by total reads number and log2 transformed.
Differentially expressed circRNAs were identified by ttest between two groups.
Genome_build: hg19
Supplementary_files_format_and_content: see headers of  CircRNA Expression Profiling.xlsx

source_name: tissue

extract_protocol:
Total RNA was extracted from the frozen kidney tissues with using TRIzol reagent
Total RNA from each sample was used to prepare the circRNA sequencing library  using standard Illumina protocols

value: lupus nephritis

tag: class

value: IV

tag: gender

value: Female


title: NGLY1Pt1i-509_OE (day 40)

value: NGLY1-functional (ectopic expression)


title: WA09-P (day 80)

value: NGLY1-functional (parental WA09)