title: CleftProband_734


title: CleftProband_730

status: Public on Dec 01 2022

type: genomic

city: Iowa City

first: J.

middle: Robert

last: Manak

state: Iowa

address: 455 Biology Building, 129 East Jefferson Street

department: Biology and Pediatrics

country: USA

institute: The University of Iowa

hyb_protocol: 34 ug of each labeled DNA was co-hybridized to a Roche NimbleGen human whole genome tiling microarray (Human CGH 2.1M Whole-Genome Tiling v2.0D Array)

scan_protocol: Arrays were scanned on a Roche NimbleGen microarray scanner.

data_processing:
532.tif and 635.tif files were analyzed using DEVA software to generate a data_summary.txt file and segMNT.txt file. Data_summary.txt files were analyzed using Nexus 7 Software to generate NexusData.bed files and then compared to the segMNT.txt file using BEDTOOLS 50% reciprocal overlap function. Only calls which shared a reciprocal overlap of 50% or greater were retained for further analysis.
BioDiscovery’s FASST2 Segmentation Algorithm, a Hidden Markov Model (HMM) based approach, was the algorithm used to make copy number calls using Nexus 7 Software. The significance threshold for segmentation was set at 1.0E-6 also requiring a minimum of 3 probes per segment and a maximum probe spacing of 1000 between adjacent probes before breaking a segment. The log ratio thresholds for single copy gain and single copy loss were set at 0.3 and -0.3, respectively. A 3:1 sex chromosome gain threshold was set to 1.2 and a 4:1 sex chromosome gain threshold was set to 1.7. NimbleGen's DEVA segMNT algorithm, which minimizes squared error relative to the segment means, was used as a second algorithm for all copy number calls after data extraction, LOESS spatial correction and background correction. Default parameters were used with the exception of setting the minimum segment difference to 0.3 and requiring a minimum of 3 probes per segment.

molecule: genomic DNA

organism: Homo sapiens

source_name: genomic DNA from whole blood

label_protocol: 1 ug of case DNA was labeled with Cy3-coupled nonamers and 1 ug of control DNA (from an unaffected male from the Philippines) was labeled with Cy5-coupled nonamers.

extract_protocol: DNA was extracted from whole blood samples using the Quickgene610L.

tag: Sex

value: male

tag: syndromic versus non-syndromic

value: non-syndromic

tag: cleft type

value: cleft lip and cleft palate

tag: cleft laterality

value: right

source_name: DNA from an unaffected male from the Philippines

value: unaffected


title: CleftProband_729

value: syndromic

value: bilateral


title: BS-Seq Idh1R132H/+ #2

type: SRA

city: New York

first: Cem

last: Meydan

state: New York

address: 1305 York

department: Physiology & Biophysics

institute: Weill Cornell Medical College

description: Idh1R132H/+ #2

library_source: genomic

data_processing:
Alignment by bwa-meth
Processing by MethylDackel
Assembly: mm10

organism: Mus musculus

source_name: Lin-cKit+Sca1+  cells

extract_protocol: To isolate LSK cells, the femur, tibia and humerus bones were dissected from mice with genotypes of interest. The bone marrow cells were isolated by flushing in 5 mL MACS buffer (phosphate-buffered saline without MgCl2 and CaCl2, supplemented with 0.5% bovine serum albumin and 2mM EDTA pH8.0) using syringes with 26 gauge needles. The isolated cells were filtered through a 70 µm nylon mesh. The cells were centrifuged at 300 x g for 5 minutes at 4°C, and resuspended in 1 mL red blood cell lysis buffer (Sigma) followed by incubation for 5 minutes at ambient temperature. 10 mL cold buffer was added, the cells were centrifuged at 300 x g for 5 minutes a 4°C, and resuspended in 1 mL cold buffer prior to counting on a ViCell Counter. The cells were then washed with 5 mL buffer, centrifuged at 300 x g for 5 minutes a 4°C, and process for lineage depletion using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec # 130-090-858), following the manufacturer’s instructions: cells were resuspended at 2.5 x 108 cells/mL in MACS buffer, and 10 µl of biotinylated mouse lineage antibody cocktail was added per 10 million cells, followed by incubation on ice for 15 minutes. 30 µl of MACS buffer and 20 µl of streptavidin-conjugated magnetic microbeads were added per 10 million cells, followed by incubation ice for 15 minutes, and filtering through a 30 µm nylon mesh in 10 mL cold MACS buffer. The cells were centrifuged at 300 x g for 5 minutes at 4°C, resuspended in 1 mL cold MACS buffer, and processed for lineage separation using an AutoMACS Pro instrument (Miltenyi Biotec) using the “Deplete” settings. Lineage-negative cells were collected.  Cells were incubated for 15 minutes on ice with MACS buffer containing 1:100 Fc blocking antibody (Tonbo Biosciences # 70-0161-M001). Cells were stained with the following antibodies in FACS or MACS buffer for 30 minutes on ice: cKIT-PE/Cy7 (BioLegend # 105814; 1:100), SCA1-FITC (BioLegend # 122505, 1:100), STREPTAVIDIN- APC/Cy7 (BioLegend # 405208; 1:50), alongside DAPI (2ug/mL). After staining, cells were washed twice with cold buffer, resuspended, and sorted using a FACSAria II fluorescence-activated cell sorter (BD Biosciences) into MACS buffer at 4°C. 1 x 105 cells were processed for RNA and DNA extraction using the AllPrep DNA/RNA Micro Kit (Qiagen #80284), following the manufacturer’s instructions.

tag: strain

tag: tissue

value: Bone marrow

tag: genotype


title: NGS02465b

city: Bethesda

first: Julie

middle: E

last: Niemela

state: MD

address: 10 Center Dr.

department: Dept. of Lab. Medicine

institute: NIH Clinical center

library_source: transcriptomic

data_processing:
Demultiplexed reads were mapped to the hg19 genome using the splice-aware aligner Tophat (Trapnell et al., 2009)
Read counts were variance stabilized (normalized) using DeSeq2
Assembly: Homo sapiens (human) genome assembly GRCh37 (hg19)
Supplementary files format and content: File with raw counts for every gene and every sample

molecule: total RNA

source_name: Blood

extract_protocol:
AmpliSeq for Illumina Transcriptome Human Gene Expression panel
Targeted Sequencing Library (>20,000 human RefSeq genes)

tag: cell type

value: CD4+ T-cell blasts

value: TCF3 wild-type / TCF3 G486Lfs*4 (HI)

tag: disease state

value: affected


title: CleftProband_726

value: left


title: CleftProband_728


title: NGS02462b

description: healthy control 3

value: TCF3 wild-type / TCF3 wild-type

value: apparently healthy


title: BS-Seq WT #3

description: WT #3

value: +/+;Vav-Cre


title: CleftProband_724


title: BS-Seq WT #2

description: WT #2


title: CleftProband_725

value: female


title: NGS02462

description: healthy control 3


title: BS-Seq Idh1R132H/+ #4

description: Idh1R132H/+ #4


title: CleftProband_735


title: BS-Seq Idh1R132H/+ #1

description: Idh1R132H/+ #1


title: CleftProband_727


title: NGS02461b

description: healthy control 2


title: CleftProband_723

value: cleft lip only


title: CleftProband_721

value: male


title: CleftProband_719

value: non-syndromic


title: CleftProband_717


title: NGS02461

description: healthy control 2


title: BS-Seq WT #1

description: WT #1


title: CleftProband_720

value: cleft lip and cleft palate


title: CleftProband_722


title: ATAC-Seq Tet2-/- #4

description: Tet2-/- #4

data_processing:
Trimming by TrimGalore, alignment by BWA, filtering by Picard + Samtools
Peak calling by MACS2 (shift reads by 75bp, extend by 150bp)
Counts by FeatureCounts
Please see nfcore atacseq pipeline for more details
Assembly: mm10

extract_protocol: To isolate LSK cells, the femur, tibia and humerus bones were dissected from mice with genotypes of interest. The bone marrow cells were isolated by flushing in 5 mL MACS buffer (phosphate-buffered saline without MgCl2 and CaCl2, supplemented with 0.5% bovine serum albumin and 2mM EDTA pH8.0) using syringes with 26 gauge needles. The isolated cells were filtered through a 70 µm nylon mesh. The cells were centrifuged at 300 x g for 5 minutes at 4°C, and resuspended in 1 mL red blood cell lysis buffer (Sigma) followed by incubation for 5 minutes at ambient temperature. 10 mL cold buffer was added, the cells were centrifuged at 300 x g for 5 minutes a 4°C, and resuspended in 1 mL cold buffer prior to counting on a ViCell Counter. The cells were then washed with 5 mL buffer, centrifuged at 300 x g for 5 minutes a 4°C, and process for lineage depletion using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec # 130-090-858), following the manufacturer’s instructions: cells were resuspended at 2.5 x 108 cells/mL in MACS buffer, and 10 µl of biotinylated mouse lineage antibody cocktail was added per 10 million cells, followed by incubation on ice for 15 minutes. 30 µl of MACS buffer and 20 µl of streptavidin-conjugated magnetic microbeads were added per 10 million cells, followed by incubation ice for 15 minutes, and filtering through a 30 µm nylon mesh in 10 mL cold MACS buffer. The cells were centrifuged at 300 x g for 5 minutes at 4°C, resuspended in 1 mL cold MACS buffer, and processed for lineage separation using an AutoMACS Pro instrument (Miltenyi Biotec) using the “Deplete” settings. Lineage-negative cells were collected.  Cells were incubated for 15 minutes on ice with MACS buffer containing 1:100 Fc blocking antibody (Tonbo Biosciences # 70-0161-M001). Cells were stained with the following antibodies in FACS or MACS buffer for 30 minutes on ice: cKIT-PE/Cy7 (BioLegend # 105814; 1:100), SCA1-FITC (BioLegend # 122505, 1:100), STREPTAVIDIN- APC/Cy7 (BioLegend # 405208; 1:50), alongside DAPI (2ug/mL). After staining, cells were washed twice with cold buffer, resuspended, and sorted using a FACSAria II fluorescence-activated cell sorter (BD Biosciences) into MACS buffer at 4°C. For ATAC-seq, the OMNI-ATAC method was used: 5 x 104 cells were resuspended in 1.5 mL microtubes in 50 μl ice-cold ATAC-Resuspension Buffer (RSB – 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% Nonidet P40, 0.1% Tween-20, and 0.01% Digitonin, and incubated on ice for 3 minutes. 1 mL cold RSB was added, and the nuclei were collected by centrifugation at 500 x g for 10 minutes at 4oC. The supernatant was carefully removed by pipetting, and the nuclei resuspended in 50 μL transposition mixture (25 μL 2x TD buffer (10 mM Tris-HCl pH 7.6, 10 mM MgCl2, 20% dimethyl formamide), 2.5 μL transposase (Nextera DNA Library Prep Kit, Illumina  # FC-121-1030), 16.5 μL PBS, 0.5 μL 1% digitonin, 0.5 μL 10% Tween-20, 5 μL H2O). Transposition reactions were incubated at 37oC for 30 minutes with 1000 RPM agitation. The DNA was isolated and cleaned using the MinElute PCR purification kit (Qiagen #28004, and stored at -20oC until further processing.


title: NGS02459

description: healthy control 1


title: NGS02459b

description: healthy control 1


title: CleftProband_715

value: cleft palate only

value: not applicable


title: CleftProband_716

value: unaffected


title: CleftProband_714


title: CleftProband_713

value: bilateral


title: ATAC-Seq Tet2-/- #3

description: Tet2-/- #3

value: Bone marrow


title: ATAC-Seq Tet2-/- #2

description: Tet2-/- #2


title: ATAC-Seq Tet2-/- #1

description: Tet2-/- #1


title: CleftProband_712

value: right


title: ATAC-Seq Idh2R172K/+ #4

description: Idh2R172K/+ #4


title: CleftProband_711


title: CleftProband_710


title: ATAC-Seq Idh2R172K/+ #3

description: Idh2R172K/+ #3


title: CleftProband_709


title: CleftProband_718


title: TS138

city: Pullman

first: Michael

middle: K

last: Skinner

state: WA

address: Abelson 507

department: SBS

institute: WSU

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 GRCh38 human 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 less than 10 mapped reads averaged across all samples were removed prior to further analysis. The MEDIPS R package was then used to calculate differential coverage between  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.
Assembly: GRCh38
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 if applicable.

source_name: buccal cells

growth_protocol: Participants for this study were MZ twins recruited from the community-based Washington State Twin Registry (WSTR). Recruitment procedures and details about the WSTR have been described 34. Participants in the current study previously participated in a study of objective measures of physical activity (PA) and locations among individuals residing in the Puget Sound area around Seattle between 2012 and 2015. Follow-up data collection was conducted in 2018 and 2019, 72 pairs completed follow-up collection out of the 144 pairs who completed the baseline study. Once participants were enrolled in the study, the study coordinator sent all study materials to the participant for remote data collection.

extract_protocol:
Frozen human buccal samples were thawed for analysis. Genomic DNA from buccal samples was prepared as follows: The buccal brush was suspended in 750 μl of cell lysis solution and 3.5 µl of Proteinase K (20 mg/ml). This suspension was incubated at 55ºC for 3 hours, then vortexed and centrifuged briefly. The lysis solution was then transferred to a new 1.5 µl microcentrifuge tube. The microcentrifuge tube with the buccal brush was centrifuged again to retain any remaining solution which was combined with the transferred lysis solution. The buccal brush was discarded and 300 µl of protein precipitation solution (Promega, A795A, Madison, WI) was added to the lysis solution. The sample was incubated on ice for 15 minutes, then centrifuged at 4C for 30 minutes. The supernatant was transferred to a fresh 2 mL microcentrifuge tube and 1,000 µl ice cold isopropanol was added along with 2 µL glycoblue. This suspension was mixed thoroughly and incubated at -20 ºC overnight. The suspension was then centrifuged at 4ºC for 20 minutes, the supernatant was discarded, and the pellet was washed with 75% ethanol, then air-dried and resuspended in 100 μl H2O. DNA concentration was measured using the Nanodrop (Thermo Fisher, Waltham, MA). Methylated DNA Immunoprecipitation (MeDIP) with genomic DNA was performed as follows: individual DNA samples (2-4 ug of total DNA) were diluted to 130 μl with 1x Tris-EDTA (TE, 10 mM Tris, 1 mM EDTA) and sonicated with the Covaris M220 using the 300 bp setting. Fragment size was verified on a 2% E-gel agarose gel. The sonicated DNA was transferred from the Covaris tube to a 1.7 ml microfuge tube, and the volume was measured. The sonicated DNA was then diluted with TE buffer (10mM Tris HCl, pH7.5; 1mM EDTA) to 400 μl, heat-denatured for 10 min at 95 C, then immediately cooled on ice for 10 min. Then 100 μl of 5X IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added to the denatured sonicated DNA. The DNA-antibody mixture was incubated overnight on a rotator at 4 C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; 11201D) were pre-washed as follows: The beads were resuspended in the vial, then the appropriate volume (50 μl per sample) was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 mL 1XPBS with 0.1% BSA and 2mM EDTA) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1xIP buffer (50 mM sodium phosphate ph7.0, 700 mM NaCl, 0.25% TritonX-100) as the initial volume of beads. 50μl of beads were added to the 500μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 hours on a rotator at 4 C. After the incubation, the bead-antibody-DNA complex was washed three times with 1X IP buffer as follows: The tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then the magnetic bead antibody pellet was washed with 1xIP buffer 3 times. The washed bead antibody DNA pellet was then resuspended in 250 μl digestion buffer with 3.5 μl Proteinase K (20mg/ml). The sample was incubated for 2-3 hours on a rotator at 55 C, then 250 μl of buffered Phenol-Chloroform- Isoamylalcohol solution was added to the sample, and the tube was vortexed for 30 sec and then centrifuged at 14,000rpm for 5 min at room temperature. The aqueous supernatant was carefully removed and transferred to a fresh microfuge tube. Then 250 μl chloroform were added to the supernatant from the previous step, vortexed for 30 sec and centrifuged at 14,000rpm for 5 min at room temperature. The aqueous supernatant was removed and transferred to a fresh microfuge tube. To the supernatant 2μl of glycoblue (20mg/ml), 20μl of 5M NaCl and 500μl ethanol were added and mixed well, then precipitated in -20 C freezer for 1 hour to overnight. The precipitate was centrifuged at 14,000rpm for 20 min at 4 C and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500μl cold 70% ethanol in -20 C freezer for 15 min then centrifuged again at 14,000rpm for 5 min at 4 C and the supernatant was discarded. The tube was spun again briefly to collect residual ethanol to the bottom of the tube and as much liquid as possible was removed with gel loading tip. The pellet was air-dried at RT until it looked dry (about 5 min) then resuspended in 20μl TE. DNA concentration was measured in Qubit (Life Technologies) with ssDNA kit (Molecular Probes Q10212).
The MeDIP DNA samples (50 ng of each) were used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, CA) starting at step 1.4 of the manufacturer’s protocol to generate double stranded DNA. After this step the manufacturer’s protocol was followed. Each sample received a separate index primer. NGS was performed at WSU Spokane Genomics Core using the Illumina HiSeq 2500 with a PE50 application, with a read size of approximately 50bp and approximately 10-35 million reads per sample, and 6-10 sample libraries each were run in one lane.

treatment_protocol: Participants wore a Qstarz BT-Q1000XT (Qstarz International Co. Ltd, Taipei, Taiwan) GPS data logger and Actigraph GT3X+ accelerometer (Actigraph Inc. Pensacola, FL) attached to a belt worn around the waist for one week. They also completed questionnaires and provided a buccal sample, with 70 complete pairs provided buccal samples. The buccal cell collection procedure involved 30 second brushing of each cheek before putting in enclosed vial for shipment by mail and then stored at -80°C. Materials were sent to the Skinner lab at WSU Pullman after sample and data collection was completed. Buccal swab brushes were stored at -80°C until use. The study protocol was approved by the Washington State University Institutional Review Board (#16419), and informed written consent was obtained from all participants prior to receiving the study materials. All methods were carried out in accordance with relevant guidelines and regulations.

value: buccal cells

tag: gender

tag: srbmi

tag: walk score

tag: mvpawk

tag: twin pair number


title: TS139


title: ATAC-Seq Idh2R172K/+ #2

description: Idh2R172K/+ #2


title: TS140


title: CleftProband_708


title: CleftProband_707


title: ATAC-Seq Idh2R172K/+ #1

description: Idh2R172K/+ #1


title: CleftProband_706


title: CleftProband_704

value: unknown


title: ATAC-Seq Idh2R140Q/+ #3

description: Idh2R140Q/+ #3


title: CleftProband_703


title: TS136


title: ATAC-Seq Idh2R140Q/+ #2

description: Idh2R140Q/+ #2


title: TS135


title: TS134


title: CleftProband_701


title: CleftProband_702


title: TS133


title: ATAC-Seq Idh2R140Q/+ #1

description: Idh2R140Q/+ #1


title: CleftProband_699


title: CleftProband_698

value: left


title: TS132


title: CleftProband_697


title: ATAC-Seq Idh1R132H/+ #4

description: Idh1R132H/+ #4


title: TS137


title: CleftProband_696

value: syndromic


title: ATAC-Seq Idh2R140Q/+ #4

description: Idh2R140Q/+ #4


title: TS131


title: CleftProband_705


title: ATAC-Seq Idh1R132H/+ #3

description: Idh1R132H/+ #3


title: CleftProband_694


title: CleftProband_695


title: CleftProband_693


title: ATAC-Seq Idh1R132H/+ #2

description: Idh1R132H/+ #2


title: TS130


title: CleftProband_691


title: CleftProband_692


title: TS129


title: CleftProband_690


title: CleftProband_700


title: TS128

description: results.walk.female.csv.gz

tag: walkability category

value: low


title: TS127

description: results.walk.female.csv.gz

value: high


title: ATAC-Seq WT #4

description: WT #4

value: +/+;Vav-Cre


title: ATAC-Seq Idh1R132H/+ #1

description: Idh1R132H/+ #1


title: ATAC-Seq WT #3

description: WT #3


title: CleftProband_685

value: cleft lip only


title: CleftProband_686

value: female

value: male


title: CleftProband_684

value: non-syndromic

value: cleft lip and cleft palate


title: ATAC-Seq WT #2

description: WT #2


title: TS125

description: results.act.male.csv.gz

tag: activity category


title: TS126

description: results.act.male.csv.gz


title: CleftProband_683


title: ATAC-Seq WT #1

description: WT #1


title: TS124


title: CleftProband_682


title: CleftProband_688


title: CleftProband_689


title: CleftProband_680


title: CleftProband_681


title: TS123


title: CleftProband_676

value: cleft palate only

value: not applicable


title: CleftProband_687

value: unaffected


title: CleftProband_677


title: CleftProband_675


title: TS121

description: results.bmi.male.csv.gz

tag: bmi category


title: TS122

description: results.bmi.male.csv.gz


title: CleftProband_674

tag: Sex


title: BulkATACseq_Control_rep_2

city: Nijmegen

first: Maria

middle: Victoria

last: Luna Velez

state: Gelderland

address: Geert Grooteplein Zuid, 30, Route 274

department: Molecular Biology

country: Netherlands

institute: Radboud University

description:
CVENRD6-2-ATAC
Organoids in ENR medium for 6 days; experiment 2

data_processing:
Preprocessing of reads was done automatically with atac-seq workflow from the seq2science pipeline v.0.3.1 (Van der Sande, Frölich, Smits, & van Heeringen, 2021). In short, reads were trimmed using fastp v0.20.1 (Chen, Zhou, Chen, & Gu, 2018) and aligned to the mouse mm10 genome using bwa-mem v0.7.17 (Li, 2013). Mitochondrial reads, multipmapper reads, mapped reads below a minimum mapping quality of 20 or reads inside the ENCODE blacklist (Amemiya et al. 2019) were filtered out. Reads were shifted +4 bp/–5 bp on the positive/negative strand to account for the tn5 inserting bias. Duplicate reads were removed with picard MarkDuplicates v2.21.2 (Picard, 2019). Peaks were called using macs2 v2.2.7 (Y. Zhang et al., 2008), with the settings --shift -100 --extsize 200 --nomodel --keep-dup 1 --buffer-size 10000 in BAM mode. Being paired-end reads, the option macs2_keep_mates was set to true. Normalization and log2-transformation of data were done using the DESeq2 package v1.34.0 (Love et al., 2014).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Processed ATAC-seq data is in two files: One file is a tab-delimited file with all identified peak locations per replicate (sample), and one with the DESeq2 normalized and log2-transformed readcounts per sample, per peak.
Supplementary_files_format_and_content: BulkATACseq_raw_counts.txt: Tab-delimited file with all identified peak locations per replicate (sample).
Supplementary_files_format_and_content: BulkATACseq_normalized_counts.txt: Tab-delimited file with DESeq2 normalized and log2-transformed readcounts per sample, per peak.

source_name: mouse small intestinal organoid

growth_protocol: Small intestinal organoids derived from a female Lgr5GFPDTR/+ mouse (Tian et al., 2011) were cultured as described previously (Sato et al., 2009). Briefly, organoids were grown embedded in a mix of 90% ice-cold RGF BME Type 2 PathClear (Cultrex, R&D Systems) and 10% Dulbecco’s modified Eagle’s Medium/Ham’s F12 (Gibco), in a humidified atmosphere at 37°C and 5% CO2. For maintenance and control conditions organoids grew in ENR medium which contained advanced Dulbecco’s modified Eagles Medium/F12 (Gibco) supplemented with 1x Penicillin‐Streptomycin (Gibco), 10 mM HEPES (Gibco), and 1x GlutaMAX Supplement (Gibco), 1x B27 (Gibco), 1.25 mM N‐acetylcysteine (Sigma Aldrich), 50 ng/mL mEGF (Gibco), 10% final volume NOGGIN conditioned medium, 5% final volume R‐SPONDIN1 conditioned medium. For maintenance, organoids were split by mechanical dissociation using a Pasteur pipette every 5 days and the culture medium was refreshed every other day (Sato et al., 2009).

extract_protocol:
Cells were pelleted and resuspended in Recovery Cell Culture Freezing Medium (GIBCO) at -80°C to maintain native chromatin. At the start of the experiment, cells were thawed quickly at 37°C and washed twice with PBS.
ATAC-seq libraries were prepared following an adapted protocol from Buenrostro et al., 2013 and Corces M.R., 2017. In brief, 50,000 cells per replicate were lysed in 50 µl of ice-cold ATAC Resuspension Buffer (RSB) containing 0.1% IGEPAL (Sigma), 0.1% Tween-20 (Sigma), and 0.01% Digitonin (Sigma) for 3 minutes on ice. Lysates were resuspended with 1 mL of cold ATAC-RSB containing 0.1% Tween-20 (Sigma) and nuclei were pelleted by centrifugation at 500 rcf for 10 minutes at 4°C in a fixed angle centrifuge. Nuclei were tagmented for 6 minutes at 37°C with in-house made Tn5 enzyme while shaking at 650 rpm. The tagmentation reaction was stopped with stop buffer (44 mM EDTA, 131 mM NaCl, 0.3% SDS, and 600 µg/ml proteinase K as final concentration). Genomic DNA was purified using 2x AMPure XP beads (Beckman Coulter) and subsequently amplified by PCR using KAPA HiFi Hotstart Ready Mix and Nextera Index Kit (Illumina) primers. Lastly, a reverse-phase 0.65x AMPure XP beads (Beckman Coulter) DNA purification step was done, followed by 1.5x AMPure XP beads (Beckman Coulter) purification. Library concentration was measured using the dsDNA Fluorescence Quantification Assays (DeNovix), and library size was determined using the BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). DNA libraries were sequenced with an Illumina NextSeq 500, at a read length of 38 bp.

treatment_protocol: For microfold (M) cell enrichment, organoids were cultured in ENR supplemented with 3 µM CHIR99021 (Axon) and 1.5 mM valproic acid (Sigma) for three days, followed by one day in ENR medium. Afterwards, treatment with 100 ng/mL of recombinant mouse TRANCE (RANKL) (BioLegend) was done for 6. Organoids grown in ENR medium was used as a control. Medium was refreshed every three days.

tag: line

value: line26 wild type, Lgr5GFPDTR/+

value: small intestinal organoid

tag: treatment

value: ENR medium

tag: time point

value: Day 6

tag: assay

value: ATAC-seq


title: TS119

description: results.bmi.male.csv.gz

value: buccal cells


title: TS120

description: results.bmi.male.csv.gz

value: low


title: CleftProband_672

tag: syndromic versus non-syndromic

tag: cleft type

tag: cleft laterality

value: left


title: BulkATACseq_RANKL_rep_3

description:
MD6-3-ATAC
Treatment with RANKL for 6 days; experiment 3

tag: tissue

value: RANKL


title: BulkATACseq_Control_rep_3

description:
CVENRD6-3-ATAC
Organoids in ENR medium for 6 days; experiment 3


title: BulkATACseq_Control_rep_1

description:
CVENRD6-1-ATAC
Organoids in ENR medium for 6 days; experiment 1


title: CleftProband_669

value: bilateral


title: CleftProband_670


title: BulkATACseq_RANKL_rep_2

description:
MD6-2-ATAC
Treatment with RANKL for 6 days; experiment 2


title: CleftProband_679

value: female

value: right


title: CleftProband_678

value: male

value: syndromic

value: Midline


title: TS118

description: results.bmi.female.csv.gz

tag: cell type

tag: gender

value: high


title: CleftProband_668

value: non-syndromic

value: cleft lip and cleft palate

value: unaffected


title: BulkChIPseq_H3K27ac_Control_rep_3

description:
Control_K27ac_3
ChIPmentation with H3K27ac antibody ENR medium for 6 days; experiment 3

data_processing:
The ChIPmentation reads were processed with the chip-seq workflow from seq2science v.0.3.1 (Van der Sande, Frölich, Smits, & van Heeringen, 2021), taking the 1% input samples of either control or RANLK-treated organoids as background for peak calling. The workflow of preprocessing, alignment, deduplication and quality filtering was similar to the workflow described for the ATAC-seq analysis, but without shifting sequencing reads in alignment or during peak calling. ChIPmentation alignments, corresponding to the H3k27ac signal, were quantified using the coverage_table function from GimmeMotifs v0.16.0 (Bruse & Heeringen, 2018) with a window of 2 kb. For the ONECUT2 ChIPmentation, we used Diffbind (78) (v.3.4.11) to identify differentially bound genomic loci between RANKL- and control-treated organoids, with an FDR-cutoff of 0.05. For visualization of the results, we used the coverage_table function from GimmeMotifs to generate a table of log-transformed counts for these differential regions for RANKL and control samples.
Assembly: mm10
Supplementary files format and content: Processed ChIP-seq data is in three files: One file is a tab delimited file with all identified peak locations for H3K27ac mark per replicate (sample), one with the GimmeMotifs normalized and log-transformed readcounts per sample, per peak and one is a tab delimited file with all identified peak locations per replicate (sample), output of DiffBind.

extract_protocol:
Following harvesting, organoids were crosslinked with 1 % formaldehyde, and glycine was added to quench the reaction. After a washing step with 1x ice-cold PBS, cell pellets were snap frozen and stored at -80°C until being processed for ChIPmentation.
A total of 250.000 cells per replicate were lysed in a buffer containing 20 mM HEPES pH 7,6, 1% SDS, 1x Protease Inhibitor Cocktails (Roche). Chromatin was sheared using the medium setting of the Biorupter Pico (Diagenode) with 6 cycles (20 seconds on/30 seconds off) to achieve fragments between 200 and 600 bp. Samples were then spun at 13.000 rpm for 5 minutes at room temperature and supernatant containing fragmented chromatin was used for ChIPmentation as described by (Schmidl et al., 2015) with the modifications published in Lindeboom et al., 2018. Briefly, before starting the immunoprecipitation, we saved 1% of the input material of either control or RANKL-treated organoids and stored it at 4° C until further processing (tagmentation). Then, chromatin of each replicate was incubated with 1 µg of antibody against H3K27ac (C15410196, Diagenode) or 1 µg of antibody against ONECUT2 (Proteintech, 21916-1-AP, AB_2848180) in dilution buffer (1% Triton X-100, 1,2 mM EDTA, 16,7 mM Tris pH 8, 167 mM NaCl, 1x Protease Inhibitor Cocktail (Roche)), rotating overnight at 4°C. Antibody-bound chromatin was purified using protein A and protein G Dynabeads (Invitrogen) followed by two washing steps with increasing concentration of NaCl and one washing step with TE buffer as described before (Lindeboom et al., 2018). Tagmentation of immunoprecipitated and input chromatin was performed with in-house generated Tn5 enzyme for 10 minutes at 37°C while shaking at 550 rpm, followed by washing steps with decreasing amounts of NaCl and soap (Lindeboom et al., 2018). Lastly, samples were de-crosslinked with 20 µg of proteinase K (Sigma) in elution buffer (0,5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris pH 8) for 1 hour at 55°C and 1.000 rpm shaking followed by an overnight incubation at 65°C and 1.000 rpm shaking. The eluted, tagmented chromatin was treated with 10 µg proteinase K (Sigma) for 1 additional hour at 55°C and purified using 2x AMPure XP beads (Beckman Coulter). The generated libraries were amplified using the KAPA HiFi Hotstart Ready Mix and Nextera Index Kit 1 (i7) and 2 (i5) primers (Illumina). Amplified libraries were purified using a 0,65x AMPure XP beads (Beckman Coulter) followed by a 1,8x AMPure XP beads (Beckman Coulter) purification. Library concentration was measured using the dsDNA Fluorescence Quantification Assays (DeNovix), and library size was determined using the BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). DNA libraries were sequenced with an Illumina NextSeq 500, and 38-bp paired-end reads were generated.

value: line26 wild type, Lgr5GFPDTR/+

value: small intestinal organoid

value: ENR medium

tag: chip antibody

value: ChIP-seq


title: TS117

description: results.bmi.female.csv.gz

value: buccal cells

value: low

tag: srbmi

tag: walk score

tag: mvpawk


title: CleftProband_666

value: cleft lip only


title: CleftProband_673

value: left


title: CleftProband_667


title: TS115

description: results.bmi.female.csv.gz

value: female

tag: twin pair number


title: BulkChIPseq_H3K27ac_Control_rep_2

description:
Control_K27ac_2
ChIPmentation with H3K27ac antibody ENR medium for 6 days; experiment 2

value: Day 6


title: CleftProband_671

value: male

value: cleft palate only

value: not applicable


title: BulkChIPseq_H3K27ac_Control_rep_1

description:
Control_K27ac_1
ChIPmentation with H3K27ac antibody; ENR medium for 6 days; experiment 1


title: CleftProband_664

value: bilateral


title: BulkATACseq_RANKL_rep_1

description:
MD6-1-ATAC
Treatment with RANKL for 6 days; experiment 1

value: RANKL

value: ATAC-seq


title: CleftProband_663

value: non-syndromic

value: cleft lip and cleft palate

value: unaffected


title: CleftProband_661


title: TS114

description: results.act.male.csv.gz

tag: activity category

value: high


title: TS112

description: results.bmi.female.csv.gz

tag: bmi category


title: BulkChIPseq_H3K27ac_RANKL_rep_3

description:
Mcell_K27ac_3
ChIPmentation with H3K27ac antibody treatment with RANKL for 6 days; experiment 3

tag: line

value: line26 wild type, Lgr5GFPDTR/+

value: small intestinal organoid

value: ChIP-seq


title: CleftProband_658

tag: Sex


title: CleftProband_665


title: TS113

description: results.act.male.csv.gz

value: buccal cells

value: low


title: CleftProband_660

value: female

value: cleft lip only

value: right


title: BulkChIPseq_H3K27ac_RANKL_rep_1

description:
Mcell_K27ac_1
ChIPmentation with H3K27ac antibody; treatment with RANKL for 6 days; experiment 1

tag: treatment

tag: time point

value: Day 6


title: CleftProband_659

tag: syndromic versus non-syndromic

value: left

value: male


title: CleftProband_662

tag: cleft type


title: scRNAseq_RANKL+CSRM617_D6_2

description:
D6-1607-L5
scRNAseq; treatment with RANKL+CSRM617 day 6

data_processing:
Raw FASTQ files were demultiplexed, aligned and annotated to the mouse genome GRCm38 indexed with sequences from the ERCC ExFold RNA Spike-In Mixes (invitrogen), and counted using the kallisto|bustools wrapper kb_phython v0.26.3 (Bray N. and Melsted P.). In case of duplicate gene entries, the entry with the highest total UMI count was kept. Pre-processing and quality control was performed using the Scater package v1.10.1 (McCarthy et al. 2017). To exclude low-quality cells from further analysis, any cell with < 500 detected features, < 1.000 total transcripts, > 40% mitochondrial transcripts, or > 20% ERCC transcripts were removed from the datasets. Normalization, natural log-transformation, scaling (with “nCount” and “nFeature” as variance to regress), and clustering analysis were performed using Seurat v3.1.5 (Butler et al., 2018). We determined the 2.000 highly variable genes using the variance stabilizing transformation (VST) method. Dimensionality reduction was performed by principal component analysis (PCA), and was followed by Louvain clustering using the FindNeighbors function with the top 30 principal components (PCs) and the FindClusters function with a resolution set to 1.6 for the RANKL treatment dataset (21.099 genes across 6.636 cells), and the top 20 PCs and a resolution of 1.5 for the dataset of RANKL and CSRM617 combined treatment (9.257 genes across 1.903 cells).
Genome_build: GRCm38
Supplementary_files_format_and_content: scRNA-Seq data is in a tab-delimited file with normalized and log2-transformed transcript reads per cell.
Supplementary_files_format_and_content: scRNAseq_RANKL_raw_counts.txt: Tab-delimited file with raw readcounts per gene, per cell, from all samples (all 384-well plates from the RANKL dataset).
Supplementary_files_format_and_content: scRNAseq_RANKL_normalized_counts.txt: Tab-delimited file with normalized and log2-transformed readcounts per gene, per cell, from all samples (all 384-well plates from the RANKL dataset), calculated with Seurat.
Supplementary_files_format_and_content: scRNAseq_CSRM617_raw_counts.txt: Tab-delimited file with raw readcounts per gene, per cell, from all samples (all 384-well plates from the RANKL+CSRM617 dataset).
Supplementary_files_format_and_content: scRNAseq_CSRM617_normalized_counts.txt: Tab-delimited file with normalized and log2-transformed readcounts per gene, per cell, from all samples (all 384-well plates from the RANKL+CSRM617 dataset), calculated with Seurat.

extract_protocol:
After harvesting, organoids were dissociated using TrypLE (Gibco). Viable single cells were sorted in 384-well PCR plates (Bio-Rad) using the Becton Dickinson Aria (BD Biosciences) sorter with a 100 µm nozzle, based on forward and side scatter gating. Prior to sorting, plates were primed with oligos (5‘-3’) containing the T7 promoter, a 5’ Illumina adapter, unique molecular identifiers (UMIs), a unique cell barcode and the oligo-(dT)N for tagging of individual mRNA molecules within each cell (Gerlach et al,. 2019). Immediately after sorting, plates were centrifuged for 2 minutes at 1.200 x g and 4°C and stored at -80°C until library preparation.
Libraries were prepared using a modified CEL-Seq2 protocol (Hashimshony et al., 2016). In brief, a micro-dispenser machine Nanodrop II (BioNex) was used to dispend in each well the ERCC spike-in control RNAs (1:50.000, Invitrogen), reagents used for the reverse transcription (RT) reaction such as Superscript II (Invitrogen) and RNasein Plus (Promega), and for the second strand synthesis such as E. coli DNA polymerase and E. coli DNA ligase (New England Biolabs). Second strand synthesis was done in the presence of E. coli RNase H (Invitrogen). Double stranded cDNA from each well was pooled together and purified with AMPure XP beads (Beckman Coulter). In vitro transcription was performed overnight according to the Ambion MegaScript IVT kit protocol (Invitrogen), followed by an exonuclease digestion step with EXOI/rSAP-IT (Applied Biosystems). The amplified RNA was later fragmented and cleaned up using AMPure XP beads (Beckman Coulter). Samples were then subjected to library RT using random octamers (Gerlach et al,. 2019) and to cDNA amplification thereafter using the Phusion High-Fidelity DNA Polymerase (New England Biolabs). A forward primer sequence against the 5’ Illumina adapter (Gerlach et al,. 2019) and a reverse primer sequence against the octamers overhang incorporated during RT were added to the amplification mix. The reverse primer contained an Illumina 3’ adapter sequence and an index sequence to uniquely identify each library (NextFlex DNA barcodes, PerkinElmer). Libraries were purified and quantified using the dsDNA Fluorescence Quantification Assays (DeNovix) and fragment sizes were assessed with the BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). The libraries were sequenced on an Illumina NextSeq 500, and 63-bp paired-end reads were generated.

treatment_protocol: For microfold (M) cell enrichment, organoids were cultured in ENR supplemented with 3 µM CHIR99021 (Axon) and 1.5 mM valproic acid (Sigma) for three days, followed by one day in ENR medium. Afterwards, treatment with 100 ng/mL of recombinant mouse TRANCE (RANKL) (BioLegend) was done for 6 or 9 days, alone or in combination with 40 µM CSRM617 Hydrochloride (Sigma-Aldrich). Organoids grown in ENR medium was used as a control. Medium was refreshed every three days.

tag: assay

value: scRNA-seq


title: CleftProband_657

tag: cleft laterality


title: CleftProband_656


title: TS111

description: results.bmi.female.csv.gz

tag: cell type


title: CleftProband_655


title: BulkChIPseq_H3K27ac_RANKL_rep_2

description:
Mcell_K27ac_2
ChIPmentation with H3K27ac antibody treatment with RANKL for 6 days; experiment 2

tag: tissue

value: RANKL


title: scRNAseq_RANKL+CSRM617_D6_1

description:
D6-1606-L3
scRNAseq; treatment with RANKL+CSRM617 day 6


title: CleftProband_654

value: non-syndromic


title: CleftProband_653

value: bilateral


title: CleftProband_652

value: cleft lip and cleft palate

value: unaffected


title: TS109

tag: gender


title: TS110

tag: srbmi


title: CleftProband_651


title: CleftProband_649


title: TS108

tag: walk score


title: CleftProband_650


title: scRNAseq_RANKL+CSRM617_D5_2

description:
D5-1599-L2
scRNAseq; treatment with RANKL+CSRM617 day 5

value: line26 wild type, Lgr5GFPDTR/+

value: Day 5


title: scRNAseq_RANKL+CSRM617_D5_1

description:
D5-1577-L5
scRNAseq; treatment with RANKL+CSRM617 day 5

value: small intestinal organoid


title: scRNAseq_RANKL+CSRM617_D4_2

description:
D4-1598-L5
scRNAseq; treatment with RANKL+CSRM617 day 4

value: Day 4


title: CleftProband_648


title: CleftProband_644


title: CleftProband_646

value: syndromic


title: CleftProband_647

value: female


title: TS105

value: buccal cells

tag: mvpawk


title: TS106

tag: twin pair number


title: CleftProband_643

value: cleft lip only


title: TS104


title: CleftProband_641


title: scRNAseq_RANKL+CSRM617_D3_2

description:
D3-1594-L1
scRNAseq; treatment with RANKL+CSRM617 day 3

value: Day 3


title: scRNAseq_RANKL+CSRM617_D3_1

description:
D3-1569-L4
scRNAseq; treatment with RANKL+CSRM617 day 3


title: CleftProband_640

value: left


title: scRNAseq_RANKL+CSRM617_D2_2

description:
D2-1596-L1
scRNAseq; treatment with RANKL+CSRM617 day 2

value: Day 2


title: TS116

description: results.bmi.female.csv.gz

value: low


title: CleftProband_642

value: male

value: right


title: CleftProband_645

value: non-syndromic


title: scRNAseq_RANKL+CSRM617_D4_1

description:
D4-1597-L2
scRNAseq; treatment with RANKL+CSRM617 day 4

value: scRNA-seq


title: TS103


title: TS102

description: results.act.female.csv.gz

tag: activity category

value: high


title: CleftProband_638

value: cleft palate only

value: not applicable

value: unaffected


title: CleftProband_639

value: cleft lip and cleft palate


title: CleftProband_637

value: bilateral


title: CleftProband_636

value: female


title: TS101

description: results.act.female.csv.gz

value: buccal cells


title: TS107


title: scRNAseq_RANKL+CSRM617_D1_2

description:
D1-1593-L3
scRNAseq; treatment with RANKL+CSRM617 day 1

value: line26 wild type, Lgr5GFPDTR/+

value: small intestinal organoid

value: Day 1


title: CleftProband_634


title: CleftProband_631

value: cleft lip only

value: left


title: CleftProband_633

value: male

value: unknown


title: CleftProband_630

value: non-syndromic


title: CleftProband_635


title: TS100


title: TS99


title: TS98

description: results.walk.female.csv.gz

tag: walkability category


title: scRNAseq_RANKL+CSRM617_D0_2

description:
D0-1591-L3
scRNAseq; treatment with RANKL+CSRM617 day 0

tag: line

value: Day 0

value: scRNA-seq


title: scRNAseq_RANKL+CSRM617_D1_1

description:
D1-1592-L1
scRNAseq; treatment with RANKL+CSRM617 day 1


title: TS97

description: results.walk.female.csv.gz

value: low


title: CleftProband_627

tag: Sex

value: cleft palate only

value: not applicable


title: scRNAseq_RANKL+CSRM617_D2_1

description:
D2-1595-L4
scRNAseq; treatment with RANKL+CSRM617 day 2

tag: treatment

value: Day 2


title: CleftProband_626

value: cleft lip and cleft palate

value: unaffected


title: CleftProband_628

value: female

value: bilateral


title: scRNAseq_RANKL+CSRM617_D0_1

description:
D0-1590-L1
scRNAseq; treatment with RANKL+CSRM617 day 0

value: line26 wild type, Lgr5GFPDTR/+

value: small intestinal organoid

tag: time point


title: scRNAseq_RANKL_D9_3

description:
INV_1_24_merged
scRNAseq; treatment with RANKL day 9

value: RANKL

value: Day 9


title: CleftProband_625

value: male

tag: syndromic versus non-syndromic


title: scRNAseq_RANKL_D9_2

description:
INV_1_12_merged
scRNAseq; treatment with RANKL day 9


title: CleftProband_624

tag: cleft type

value: right


title: TS96

description: results.bmi.female.csv.gz;results.act.female.csv.gz

value: buccal cells

tag: bmi category

value: high


title: scRNAseq_RANKL_D9_1

description:
INV_1_03_merged
scRNAseq; treatment with RANKL day 9

tag: assay

value: scRNA-seq


title: CleftProband_632

value: non-syndromic

value: cleft lip only

tag: cleft laterality


title: CleftProband_623

value: left


title: TS95

tag: cell type

value: low


title: CleftProband_621

value: cleft lip and cleft palate


title: CleftProband_629

value: unaffected


title: TS94

tag: gender

value: female


title: scRNAseq_RANKL_D8_3

description:
INV_1_26_merged
scRNAseq; treatment with RANKL day 8

tag: tissue

value: Day 8


title: CleftProband_619


title: scRNAseq_RANKL_D8_2

description:
INV_1_16_merged
scRNAseq; treatment with RANKL day 8

value: line26 wild type, Lgr5GFPDTR/+


title: CleftProband_620


title: TS93

tag: srbmi

tag: walk score


title: scRNAseq_RANKL_D8_1

description:
INV_1_04_merged
scRNAseq; treatment with RANKL day 8

value: small intestinal organoid


title: CleftProband_617


title: CleftProband_618

value: bilateral


title: scRNAseq_RANKL_D7_3

description:
INV_1_23_merged
scRNAseq; treatment with RANKL day 7

value: RANKL

value: Day 7


title: TS92

tag: mvpawk

tag: twin pair number


title: TS91


title: CleftProband_616

value: right

value: male


title: CleftProband_615


title: CleftProband_622


title: scRNAseq_RANKL_D7_2

description:
INV_1_15_merged
scRNAseq; treatment with RANKL day 7


title: CleftProband_614

value: syndromic


title: CleftProband_613


title: CleftProband_612


title: CleftProband_610


title: scRNAseq_RANKL_D6_3

description:
INV_1_30_merged
scRNAseq; treatment with RANKL day 6

value: Day 6

value: scRNA-seq


title: TS90

description: results.bmi.male.csv.gz

value: buccal cells

value: high


title: TS89

description: results.bmi.male.csv.gz

value: low


title: CleftProband_609

value: non-syndromic

value: cleft lip and cleft palate

value: left


title: CleftProband_611

value: female

value: cleft lip only

value: unaffected


title: TS88


title: scRNAseq_RANKL_D6_2

description:
INV_1_17_merged
scRNAseq; treatment with RANKL day 6

value: line26 wild type, Lgr5GFPDTR/+

value: small intestinal organoid


title: TS87


title: CleftProband_608


title: CleftProband_607


title: scRNAseq_RANKL_D6_1

description:
INV_1_02_merged
scRNAseq; treatment with RANKL day 6

value: RANKL


title: CleftProband_606

value: right


title: CleftProband_605

value: cleft palate only

value: not applicable

value: male


title: CleftProband_604


title: scRNAseq_RANKL_D5_3

description:
INV_1_29_merged
scRNAseq; treatment with RANKL day 5

value: Day 5

value: scRNA-seq


title: TS85

value: buccal cells


title: TS86


title: scRNAseq_RANKL_D7_1

description:
INV_1_01_merged
scRNAseq; treatment with RANKL day 7

value: Day 7


title: CleftProband_602

value: non-syndromic

value: bilateral


title: TS84

description: results.act.female.csv.gz

tag: activity category

value: low


title: scRNAseq_RANKL_D5_2

description:
INV_1_09_merged
scRNAseq; treatment with RANKL day 5

tag: line


title: CleftProband_601

tag: Sex

value: female

value: cleft lip and cleft palate

value: left


title: scRNAseq_RANKL_D5_1

description:
INV_1_05_merged
scRNAseq; treatment with RANKL day 5

value: line26 wild type, Lgr5GFPDTR/+

tag: treatment


title: TS83

description: results.act.female.csv.gz

value: high


title: CleftProband_599

value: unaffected


title: TS82

description: results.walk.male.csv.gz;results.act.female.csv.gz

tag: walkability category


title: TS81


title: CleftProband_598

tag: syndromic versus non-syndromic


title: scRNAseq_RANKL_D4_3

description:
INV_1_27_merged
scRNAseq; treatment with RANKL day 4

value: small intestinal organoid

value: RANKL

tag: time point

value: Day 4


title: CleftProband_600

value: syndromic

tag: cleft type

value: male


title: CleftProband_603


title: scRNAseq_RANKL_D4_1

description:
INV_1_06_merged
scRNAseq; treatment with RANKL day 4

tag: assay

value: scRNA-seq


title: CleftProband_597

tag: cleft laterality

value: unknown


title: CleftProband_595

value: cleft lip only


title: scRNAseq_RANKL_D4_2

description:
INV_1_13_merged
scRNAseq; treatment with RANKL day 4


title: CleftProband_596

value: non-syndromic


title: CleftProband_594

value: bilateral


title: TS80

description: results.walk.male.csv.gz

tag: cell type

value: buccal cells


title: CleftProband_593


title: TS79

description: results.walk.male.csv.gz

tag: gender

value: low


title: CleftProband_592

value: cleft lip and cleft palate

value: right


title: scRNAseq_RANKL_D3_2

description:
INV_1_07_merged
scRNAseq; treatment with RANKL day 3

value: line26 wild type, Lgr5GFPDTR/+

tag: tissue

value: Day 3


title: CleftProband_590

value: female

value: left

value: unaffected


title: CleftProband_589


title: TS78

description: results.bmi.male.csv.gz

tag: bmi category

value: high

tag: srbmi


title: CleftProband_591


title: CleftProband_588


title: TS77

description: results.bmi.male.csv.gz

tag: walk score

tag: mvpawk


title: scRNAseq_RANKL_D3_1

description:
INV_1_21_merged
scRNAseq; treatment with RANKL day 3

value: small intestinal organoid

value: RANKL


title: CleftProband_587

value: male


title: CleftProband_586


title: scRNAseq_RANKL_D2_3

description:
INV_1_28_merged
scRNAseq; treatment with RANKL day 2

value: Day 2

value: scRNA-seq


title: CleftProband_583

value: non-syndromic

value: cleft palate only

value: not applicable


title: scRNAseq_RANKL_D2_2

description:
INV_1_20_merged
scRNAseq; treatment with RANKL day 2


title: CleftProband_584


title: TS76

description: results.bmi.female.csv.gz;results.act.female.csv.gz

value: buccal cells

value: low

tag: activity category

tag: twin pair number


title: CleftProband_581

tag: Sex


title: CleftProband_582

value: cleft lip and cleft palate


title: scRNAseq_RANKL_D2_1

description:
INV_1_08_merged
scRNAseq; treatment with RANKL day 2

tag: line

value: line26 wild type, Lgr5GFPDTR/+

tag: treatment


title: CleftProband_580

tag: syndromic versus non-syndromic

value: syndromic

value: right


title: CleftProband_578

value: female

value: left


title: TS73

description: results.act.male.csv.gz


title: CleftProband_579

value: unaffected


title: TS75

value: high


title: TS74

description: results.act.male.csv.gz


title: scRNAseq_RANKL_D1_2

description:
INV_1_19_merged
scRNAseq; treatment with RANKL day 1

value: small intestinal organoid

value: RANKL

tag: time point

value: Day 1


title: TS72

description: results.walk.female.csv.gz

tag: walkability category


title: scRNAseq_RANKL_D1_3

description:
INV_1_11_merged
scRNAseq; treatment with RANKL day 1

tag: assay

value: scRNA-seq


title: CleftProband_577

value: male

value: non-syndromic

tag: cleft type

tag: cleft laterality

value: bilateral


title: scRNAseq_RANKL_D3_3

description:
INV_1_18_merged
scRNAseq; treatment with RANKL day 3

value: Day 3


title: scRNAseq_RANKL_D1_1

description:
INV_1_25_merged
scRNAseq; treatment with RANKL day 1


title: CleftProband_575


title: CleftProband_585


title: TS70

description: results.bmi.female.csv.gz

tag: cell type

value: buccal cells

tag: gender


title: CleftProband_573

value: cleft lip only


title: CleftProband_572

value: cleft lip and cleft palate


title: CleftProband_574


title: TS69

description: results.bmi.female.csv.gz

tag: bmi category

value: low


title: scRNAseq_RANKL_D0_3

description:
INV_1_22_merged
scRNAseq; treatment with RANKL day 0

value: line26 wild type, Lgr5GFPDTR/+

tag: tissue

value: Day 0


title: scRNAseq_RANKL_D0_2

description:
INV_1_14_merged
scRNAseq; treatment with RANKL day 0


title: CleftProband_571

value: female

value: right


title: TS68

tag: srbmi

tag: walk score


title: CleftProband_568

value: Midline

value: unaffected


title: CleftProband_576


title: CleftProband_570

value: cleft palate only

value: not applicable


title: CleftProband_567


title: BulkRNAseq_RANKL+CSRM617_rep_3

description:
RankLOC2in_D6_3_A84
BulkRNAseq; treatment with RANKL+CSRM617 for 6 days; experiment 3

data_processing:
Paired-end reads were aligned to the mouse mm10 genome using the STAR RNAseq-aligner v2.7.9a (Dobin et al. 2013), with –sjdbOverhang set to 100. Mapped reads were counted with HTSeq-count v0.13.5 (Anders et al., 2015) and the following settings: -r pos –s reverse –t exon. Normalization and log2-transformation of data was done using the DESeq2 package v1.34.0 (Love et al., 2014).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Processed RNA-Seq data has two files: One tab-delimited file with raw readcounts per gene, per replicate (sample), and another tab-delimited file with DESeq2 normalized and log2-transformed readcounts per gene, per sample.
Supplementary_files_format_and_content: BulkRNAseq_raw_counts.txt: Tab-delimited file with raw readcounts per gene, per replicate (sample).
Supplementary_files_format_and_content: BulkRNAseq_normalized_counts.txt: Tab-delimited file with DESeq2 normalized and log2-transformed readcounts per gene, per replicate (sample).
Supplementary_files_format_and_content: BulkRNAseq_RANKL+CSRM617_raw_counts.txt: Tab-delimited file with raw readcounts per gene, per replicate (sample).
Supplementary_files_format_and_content: BulkRNAseq_RANKL+CSRM617_normalized_counts.txt: Tab-delimited file with DESeq2 normalized and log2-transformed readcounts per gene, per replicate (sample).

extract_protocol:
Pellets were snap frozen and stored at ‐80 ⁰C. RNA from snap frozen pellets of drug treated organoids was extracted using the RNeasy RNA extraction kit (Qiagen) with DNaseI treatment.
Total RNA (1 µg per replicate) was used for library preparations using the KAPA RiboErase Kit (HMR) (Roche) following manufacturer’s protocol. A fragmentation step was carried out for 6.5 minutes at 94°C and 7 μM NextFlex DNA barcodes (PerkinElmer) were used for adapter ligation. Libraries were amplified using 6 amplification cycles. Library concentration was measured using the dsDNA Fluorescence Quantification Assays (DeNovix), and library size was determined using the BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies) in a BioAnalyzer instrument (Agilent). Sequencing was performed using an Illumina NextSeq 500, and 42-bp paired-end reads were generated.

value: small intestinal organoid

value: Day 6

value: RNA-seq


title: CleftProband_565

value: syndromic

value: left

value: male


title: CleftProband_566

value: non-syndromic

value: cleft lip only


title: scRNAseq_RANKL_D0_1

description:
INV_1_10_merged
scRNAseq; treatment with RANKL day 0

value: RANKL

value: scRNA-seq


title: TS71

description: results.walk.female.csv.gz

value: buccal cells

value: low

tag: mvpawk


title: TS66

description: results.bmi.male.csv.gz

value: high

tag: twin pair number


title: CleftProband_564

tag: Sex

value: female

value: cleft lip and cleft palate


title: CleftProband_563

value: right

value: unaffected


title: TS67


title: CleftProband_562

value: bilateral


title: BulkRNAseq_RANKL+CSRM617_rep_2

description:
RankLOC2in_D6_2_A83
BulkRNAseq; treatment with RANKL+CSRM617 for 6 days; experiment 2

tag: line

value: line26 wild type, Lgr5GFPDTR/+

value: small intestinal organoid

value: Day 6


title: TS64

description: results.walk.female.csv.gz


title: BulkRNAseq_RANKL+CSRM617_rep_1

description:
RankLOC2in_D6_1_A82
BulkRNAseq; treatment with RANKL+CSRM617 for 6 days; experiment 1

tag: treatment

value: RNA-seq


title: BulkRNAseq_RANKL_II_rep_3

description:
RankL_D6_3_A81
BulkRNAseq; treatment with RANKL (for experiments with CSRM617) for 6 days; experiment 3


title: CleftProband_561

tag: syndromic versus non-syndromic

value: cleft palate only

value: not applicable

value: male


title: BulkRNAseq_RANKL_II_rep_2

description:
RankL_D6_2_A80
BulkRNAseq; treatment with RANKL (for experiments with CSRM617) for 6 days; experiment 2

value: RANKL

tag: time point


title: TS63

description: results.walk.female.csv.gz

tag: walkability category


title: CleftProband_560

value: non-syndromic


title: CleftProband_559

value: cleft lip only

value: left


title: CleftProband_558


title: BulkRNAseq_RANKL_II_rep_1

description:
RankL_D6_1_A79
BulkRNAseq; treatment with RANKL (for experiments with CSRM617) for 6 days; experiment 1

tag: assay


title: TS62

description: results.act.male.csv.gz

value: buccal cells

tag: activity category

value: high


title: CleftProband_569

tag: cleft type

value: cleft lip and cleft palate

tag: cleft laterality


title: CleftProband_556

value: right


title: TS61

description: results.act.male.csv.gz

tag: cell type

tag: gender

value: low


title: CleftProband_555

value: bilateral

value: unaffected


title: TS65

description: results.bmi.male.csv.gz

tag: bmi category


title: CleftProband_553


title: CleftProband_557

value: female


title: BulkRNAseq_Control_rep_3

description:
CVENR_MSIO_D6_3
BulkRNAseq; ENR medium for 6 days; experiment 3

value: line26 wild type, Lgr5GFPDTR/+

tag: tissue

value: small intestinal organoid

value: ENR medium

value: Day 6


title: CleftProband_552

value: male

value: unknown


title: BulkRNAseq_Control_rep_1

description:
CVENR_MSIO_D6_1
BulkRNAseq; ENR medium for 6 days; experiment 1

value: RNA-seq


title: TS59

description: results.act.female.csv.gz

tag: srbmi

tag: walk score


title: BulkRNAseq_Control_rep_2

description:
CVENR_MSIO_D6_2
BulkRNAseq; ENR medium for 6 days; experiment 2


title: CleftProband_554

value: non-syndromic


title: TS60

description: results.act.female.csv.gz

tag: mvpawk

tag: twin pair number


title: BulkRNAseq_RANKL_rep_3

description:
Mcells_MSIO_D6_3
BulkRNAseq; treatment with RANKL for 6 days; experiment 3

tag: line

value: RANKL


title: CleftProband_549

tag: Sex

value: cleft lip only

value: left


title: TS58

description: results.bmi.female.csv.gz;results.act.female.csv.gz

value: buccal cells

value: high


title: CleftProband_548

tag: syndromic versus non-syndromic

value: cleft palate only

value: not applicable


title: TS57

value: low


title: BulkRNAseq_RANKL_rep_1

description:
Mcells_MSIO_D6_1
BulkRNAseq; treatment with RANKL for 6 days; experiment 1

tag: treatment

tag: time point


title: CleftProband_547

value: female

value: cleft lip and cleft palate

value: right

value: unaffected


title: CleftProband_545


title: TS56


title: CleftProband_544

value: bilateral


title: CleftProband_543

value: male


title: ATAC-seq_CM Mettl3-KO_rep3

city: Hangzhou

first: Yuanhui

last: Mao

state: Zhejiang

address: 1369 West Wenyi Road

country: China

institute: Zhejiang University

description: Chromatin state in Mettl3-KO differentiated CMs

data_processing:
The sequencing adaptors were removed by Cutadapt.
For ATAC-seq, the trimmed reads were aligned to mouse genome using Bowtie. The PCR duplicates were removed using Picard. Peak calling was done by MACS2.
For RNA-seq, the trimmed reads were aligned using HISAT2. Reads on individual genes and m6A peak calling were done using house-hold Perl scripts.
Genome_build: mm10
Supplementary_files_format_and_content: For ATAC-seq, the processed data files are bed-format files containing all peaks identified by MACS2. For RNA-seq, the processed data files are RPKM table. The first coloum is gene name, the second coloum is RPKM value. For m6A-seq, the processed data files are m6A peaks.  The first coloumn is gene name, the second coloum is the position of m6A motif, the third coloumn is the m6A motif.

source_name: Differentiated cardiomyocyte

growth_protocol: ESCs were cultured the medium containing DMEM, 15% fetal bovine serum, 1% penicillin/streptomycin, 1% GlutaMax  and 1 mM ascorbic acid to induce embryoid body (EB) formation. EBs were resuspended in fresh cardiomyocyte differentiation medium and replated on 0.2% gelatin coated dishes to trigger cardiomyocyte formation.

extract_protocol:
Cells were trypsinized, washed, and resuspended in ice cold PBS. ATAC-seq was performed on 100,000 cells with the following modifications: The transposition reaction was inactivated by adding 200 μl SDS Buffer (100 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA, 0.5% SDS, 100 μg Proteinase K) at 55°C for 30 minutes followed by isopropanol precipitation. The pellet was resuspended in 10 mM Tris-HCl, pH 8.0. Libraries were size-selected using Ampure XP beads (large fragments were removed using a 0.5× selection followed by a 1.8× selection) followed by PCR on half the product using Q5 High-Fidelity DNA Polymerase (New England Biolabs), Program: 98°C 30 s, 10 cycles of (98°C 10 s; 63°C 30 s; 72°C 1 min), 72°C 1 min. Amplified libraries were purified using Ampure XP beads (1.8×). Single end sequencing was performed using 75 bp reads on a NextSeq 500.
For ATAC-seq, cells were trypsinized, washed, and resuspended in ice cold PBS. ATAC-seq was performed on 100,000 cells with the following modifications: The transposition reaction was inactivated by adding 200 μl SDS Buffer (100 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA, 0.5% SDS, 100 μg Proteinase K) at 55°C for 30 minutes followed by isopropanol precipitation. The pellet was resuspended in 10 mM Tris-HCl, pH 8.0. Libraries were size-selected using Ampure XP beads (large fragments were removed using a 0.5× selection followed by a 1.8× selection) followed by PCR on half the product using Q5 High-Fidelity DNA Polymerase (New England Biolabs), Program: 98°C 30 s, 10 cycles of (98°C 10 s; 63°C 30 s; 72°C 1 min), 72°C 1 min. Amplified libraries were purified using Ampure XP beads (1.8×). Single end sequencing was performed using 75 bp reads on a NextSeq 500. For RNA-seq, RNAs associated with chromatin were purified and fragmented. Fragmented ChrRNA or m6A-containing ChrRNA was dephosphorylation in the 15 μl mixture containing 1× T4 poly-nucleotide kinase buffer, 10 U SUPERase-In, and 20 U T4 polynucleotide kinase at 37°C for 1 h. RNA was separated on a 15% polyacrylamide TBE-urea gel (Invitrogen) and visualized using SYBR Gold (Invitrogen). Selected regions of the gel corresponding to 40-60 nucleotides (nt) were excised and disrupted by using centrifugation through the holes at the bottom of the tube. Then the gel pieces were dissolved by soaking overnight in 400 μL RNA elution buffer (300 mM NaOAc, pH 5.5, 1 mM EDTA, and 0.1 U ml−1 SUPERase-In). After removal of gel debris using Spin-X column (Corning), RNA was precipitated by one-tenth volumes of 3 M sodium acetate (pH 5.2), 100 μg ml−1 glycogen, and 3 volumes of 100% ethanol overnight. RNA pellets were precipitated by centrifuging at 15,000 rpm for 20 min and washed by 70% ethanol. Purified RNA fragments were re-suspended in nuclease-free water. Poly-(A) tailing reaction was carried out for 45 min at 37°C (1× poly-(A) polymerase buffer, 1 mM ATP, 0.75 U/ml SUPERase_ In and 3 U E. coli poly-(A) polymerase).  For reverse transcription, the primers containing barcodes were listed as follows (Ø represents an abasic residue):  MCA02, 5’-pCAGATCGTCGGACTGTAGAACTCTØCAAGCAGAAGACGGCATACGATT TTTTTTTTTTTTTTTTTTVN-3’;   LGT03, 5′-pGTGATCGTCGGACTGTAGAACTCTØCAAGCAGAAGACGGCATACGATTT TTTTTTTTTTTTTTTTTVN-3′;   YAG04, 5′-pAGGATCGTCGGACTGTAGAACTCTØCAAGCAGAAGACGGCATACGATT TTTTTTTTTTTTTTTTTTVN-3′;   HTC05, 5′-pTCGATCGTCGGACTGTAGAACTCTØCAAGCAGAAGACGGCATACGATT TTTTTTTTTTTTTTTTTTVN-3′.  The tailed-RNA sample was mixed with 0.5 mM dNTP and 2.5 mM synthesized primer and incubated at 65°C for 5 min, followed by incubation on ice for 5 min. The reaction mix was then added with 1 X SuperScript III buffer, 10mM DTT, 40 U RNaseOUT and 200 U SuperScript III and incubated at 50°C for 50 min. Reverse transcription products were separated on a 10% polyacrylamide TBE-urea gel and the corresponding region was excised as described earlier. The cDNA was recovered by using DNA gel elution buffer (300 mM NaCl, 1 mM EDTA). First-strand cDNA was circularized in a mixture containing 1× CircLigase buffer, 2.5 mM MnCl2, 1 M Betaine, and 100 U CircLigase II (Epicenter) at 60 °C for 1 h. The reaction was heat inactivated at 80°C for 10 min followed by ethanol precipitation.

tag: genotype

value: ESC-derived cardiomyocyte

tag: strain

tag: antibody


title: TS55


title: CleftProband_551

tag: cleft type

tag: cleft laterality


title: CleftProband_542

value: syndromic


title: CleftProband_550

value: non-syndromic

value: unknown


title: CleftProband_541


title: CleftProband_540


title: CleftProband_539


title: ATAC-seq_CM Mettl3-KO_rep2

tag: cell type


title: BulkRNAseq_RANKL_rep_2

description:
Mcells_MSIO_D6_2
BulkRNAseq; treatment with RANKL for 6 days; experiment 2

value: line26 wild type, Lgr5GFPDTR/+

value: small intestinal organoid

value: RANKL

value: Day 6

tag: assay

value: RNA-seq


title: ATAC-seq_CM Mettl3-KO_rep1


title: CleftProband_537


title: ATAC-seq_CM WT_rep2

description: Chromatin state in WT differentiated CMs

value: WT


title: CleftProband_536

value: left


title: CleftProband_535

value: female


title: TS54

description: results.bmi.female.csv.gz

value: buccal cells

tag: gender

tag: bmi category

value: high


title: CleftProband_538

value: cleft palate only

value: not applicable

value: unaffected


title: TS53

description: results.bmi.female.csv.gz

value: low

tag: srbmi

tag: walk score


title: CleftProband_546

value: cleft lip and cleft palate

value: male


title: CleftProband_533

value: non-syndromic

value: bilateral


title: CleftProband_534


title: TS51

description: results.act.male.csv.gz

tag: activity category

tag: mvpawk

tag: twin pair number


title: ATAC-seq_CM WT_rep1

value: ESC-derived cardiomyocyte


title: CleftProband_532

tag: Sex

tag: syndromic versus non-syndromic

value: right


title: TS50


title: TS49


title: ATAC-seq_ESC Mettl3-KO_rep3

description: Chromatin state in Mettl3-KO ESCs

source_name: Embryonic stem cell

value: ESC


title: CleftProband_531


title: CleftProband_530


title: ATAC-seq_ESC Mettl3-KO_rep2

description: Chromatin state in Mettl3-KO ESCs


title: TS48

description: results.bmi.female.csv.gz


title: TS52

description: results.act.male.csv.gz


title: ATAC-seq_CM WT_rep3


title: CleftProband_529


title: CleftProband_528


title: ATAC-seq_ESC Mettl3-KO_rep1

description: Chromatin state in Mettl3-KO ESCs


title: CleftProband_527


title: TS47

description: results.bmi.female.csv.gz


title: TS46

description: results.walk.male.csv.gz

tag: walkability category


title: CleftProband_525


title: CleftProband_523

value: cleft lip only


title: CleftProband_526


title: CleftProband_524


title: CleftProband_522


title: ATAC-seq_ESC WT_rep3

description: Chromatin state in WT ESCs

tag: genotype

value: WT


title: CleftProband_521

value: left


title: TS44

value: female


title: ATAC-seq_ESC WT_rep2

description: Chromatin state in WT ESCs

tag: strain


title: CleftProband_520


title: TS43

value: buccal cells


title: CleftProband_519


title: E15.5_2b

city: Boston

first: Jyoti

last: Rao

state: Massachusetts

address: 60 Fenwood road

department: Pathology/Genetics

institute: Brigham and Women’s Hospital/Harvard Medical School

description:
Single-cell RNA-Seq (inDrops)
processed data files:
E13.5-15.5_matrix.csv
E13.5-15.5_CellData.csv
E13.5-15.5_GeneData.csv

data_processing:
Single-cell RNA-seq data were generated using inDrop version 3. Raw sequencing FASTQ files were demultiplexed and processed using a custom python pipeline as previously described (see Zilionis et al., Nature Protocols 2017) Also available at github.com/indrops/indrops. Default parameters were applied (for Trimmomatic, LEADING: "28", SLIDINGWINDOW: "4:20", MINLEN: "16"; for UMI quantification, m: 10, u: 1, d: 600, split-ambigs: False, min_non_polyA: 15; for low complexity filter, max_low_complexity_fraction: 0.50; for output: output_unaligned_reads_to_other_fastq: False, filter_alignments_to_softmasked_regions: False).
Sequencing reads were first sorted according to sample of origin.  Each library is associated with 4 reads - R1, R2, R3 and R4.  R1 is the biological read, R2 carries the first half of the hydrogel bead barcode (8 bp), R3 carries the library index (8 bp), and R4 carries the second half of the hydrogel bead barcode (8 bp), followed by the UMI (6 bp).
Cell-specific barcodes for each cDNA read were then matched against a set of pre-determined barcodes.
cDNA reads were then aligned to a reference transcriptome using Bowtie (version 1.2.2, parameters: -n 1 -l 15 -e 100 -m 200 -best -strata -a).  Reference transcriptomes were built from Homo_sapiens.GRCh38.94 and Mus_musculus.GRCm38.94 genome assembly. Mapped reads were then processed into counts of UMI-filtered transcripts per gene.
UMI counts matrices were filtered to exclude cell barcodes associated with low numbers of reads.  This determination was made by inspecting a weighted histogram of UMI counts for each cell barcode and thresholds were set manually for each sample. We excluded the cells with less than 250 genes expressed, and exclude the genes if they were expressed in less than 3 cells. Fraction of counts due to mitochondrial genes (n) was determined and cells with n= 0.2 were removed. Cells with less than 250 genes expressed were removed. Genes expressed in less than 3 cells were filtered out. Fraction of counts due to mitochondrial genes (n) was determined and cells with n= 0.2 were removed.
Genome_build: Homo_sapiens.GRCh38.94 and Mus_musculus.GRCm38.94
Supplementary_files_format_and_content: X.csv: Raw UMI-filtered counts.  Matrix columns are transcripts, rows are single cells.
Supplementary_files_format_and_content: CellData.csv: Identifiers for single cells corresponding to the rows of X.csv.  Unique cell identifiers are in the following format: LibraryName_CellBarcodePart1_CellBarcodePart2.  Numbers of UMIS and genes detected and clustering assignments are included for each cell
Supplementary_files_format_and_content: GeneData.csv: Gene names corresponding to the columns of X.csv

molecule: polyA RNA

source_name: Mouse embryonic tissue

extract_protocol:
Single cell analysis of differentiating iPSCs and mouse embryos cells was performed using inDrops as previously reported (Klein et al.). UCP1-mCherry iPSC line was differentiated into brown adipocytes as described above. Cultures were harvested on day 6, 10, 15, 20, 30 of the primary differentiation and day 20 and 40 after replating.  The cultures were washed with PBS and dissociated into single cells using 2.5mg/ml Collagenase and 0.05% Trypsine EDTA in PBS for 5-15 minutes at 37ºC. Dissociated cells were run through 70µm cell strainer followed by 30µm cell strainer, spun at 300g for 5 minutes and resuspended in DMEM supplemented with 5% fetal bovine serum. Cells were again spun at 300g for 5 minutes and resuspended in DMEM + 5% fetal bovine serum, this was repeated 2-3 times to remove debris. Final resuspension was made in PBS + 0.1% bovine serum albumin. Cell density was adjusted to 200,000 cells/ml. For day 6, 10, 15 and 20, 2500 cells were collected from each sample. For samples 30 day, 20 day after replating and 40 day after replating, at least 3000 cells were collected from each sample from two independent differentiations. For analyzing mouse embryonic tissues, embryos were collected on embryonic day 13.5, 14.5 and 15.5 from wildtype CD1 IGS mice. For each stage, back tissue dorsal to rib cage at the level of forelimb was dissected out. Neural tube/spinal cord and dorsal root ganglion were removed wherever possible. For E15.5, epidermis and underlying dermis was removed before dissociating the tissue. Dissected tissues were washed several times in Hanks' Balanced Salt Solution. Tissues were transferred to microfuge tube with 200µl of 2.5mg/ml Collagenase and 0.05% Trypsine EDTA in PBS, chopped into small pieces using scissors and incubated at 37ºC for 15 minutes with intermittent shaking. Tissues were mechanically dissociated by triturating several times using wide-bore 1ml pipette. The resulting cell suspension was mixed with HBBS + 10% FBS, filtered through a 30µm cell strainer and spun at 300g for 5 minutes. To remove red blood cells, cell pellet was resuspended and incubated with RBC Lysis Solution for 5 minutes at room temperature. Cells were washed with HBSS + 10% FBS for 2-3 times to remove debris. Final suspension was made in PBS + 0.1% BSA. Cell density was adjusted to 200,000/ml. For each stage, cells were collected from two littermate embryos. For E13.5-14.5  embryo, 3000 cells were encapsulated and sequenced from each embryo. For E15.5, 5000 cells were collected and sequenced from each embryo.
Single-cell RNA-Seq libraries were prepared using a protocol customized for the inDrops platform (see Zilionis et al., Nature Protocols 2017)

value: embryonic tissue

value: CD1 IGS wildtype

tag: library name


title: CleftProband_517

tag: cleft type


title: TS42


title: E15.5_2a


title: CleftProband_516

tag: cleft laterality


title: E15.5_1b

tag: cell type


title: TS41


title: CleftProband_514


title: CleftProband_515


title: TS40

description: results.bmi.female.csv.gz;results.walk.female.csv.gz

value: high


title: TS45

description: results.walk.male.csv.gz


title: E15.5_1a


title: E14.5_2


title: CleftProband_513

value: unaffected


title: CleftProband_511


title: E13.5_2


title: CleftProband_507


title: TS37


title: CleftProband_510


title: CleftProband_518

value: cleft palate only

value: not applicable


title: CleftProband_506

value: male

value: cleft lip and cleft palate


title: ATAC-seq_ESC WT_rep1

description: Chromatin state in WT ESCs

tag: antibody


title: CleftProband_505

value: non-syndromic


title: CleftProband_509


title: CleftProband_508


title: Enthesis Gli1 P42

first: Fei

last: Fang

address: 650 W 168th Street, Blacking Building 1408

department: Orthopedic Surgery

institute: Columbia University

data_processing:
Raw sequencing output were processed through 10X genomics Cell Rnager software suite using default setting.
Reads were mapped to the mm10 transcriptome. Read counts were normalized to log2 reads per million mapped (rpm) and a cutoff of log2 rpm >= 1 was used to determine whether a given gene was expressed in a cell.
Cells with genes expressed less than 2 cells, cells<1000 UMIs with >20% UMIs mapped to mitochondrial genes, and <200 genes were removed.
Genome_build: mm10
Supplementary_files_format_and_content: tsv and mtx files describe barcode, features, and mattrix for each sample
Supplementary_files_format_and_content: comma-separated text files include features and matrix for each sample

source_name: Supraspinatus tendon enthesis

extract_protocol:
Tendon entheses from P11, P18, and P56 C57BL/6J mice were dissected under surgical microscope and pooled together. They were digested in HBSS solution with 300 mg/ml liberase, 20 ng/ml Dnase, 5% FBS, and 100 mg/ml HEPES for 1.5 hours at 37 celsius degree. The samples were filtered through a 40 μm strain, resuspended in the RBC lysis buffer for 5 minutes at room temperature and stained with Cd45 in 200 μl PBS with 5% FBS for 45 minutes at 4°C. Then, the samples were washed with PBS three times, resuspended in 500 μl PBS with 1 ug/ml DAPI and 5% FBS, and filtered into polypropylene FACS tubes. Single cells without Cd45 and DAPI staining were index sorted by a BD influx Sorter and collected into a microcentrifuge tube for scRNA-seq 10 Genomics experiments. For dissociation of Gli1-lineage cells, tendon enthesesof GliCreERT;Ai14 mice of P7, P11, P18, P21, P28, and P42 were dissected, pooled together, and digested in the aforementioned solution under the same condition discussed above. All cells were stained with 1 ug/ml DAPI and were index sorted by a BD influx Sorter. Single cells without DAPI staining and red fluorescence were collected into 96-well plates, prefilled with lysis buffer for scRNA-Seq analysis.
For all enthesis cells, resulting pooled, 3’-end libraries was sequenced on Illumina® NovaSeq™ 6000; For Gli1-lineage cells, Pooled 3’-end sequencing libraries were prepared and finally sequenced on an IIlumina NextSeq 500

tag: tissue

value: Tendon enthesis

tag: age

value: post natal day 42

tag: treatment

value: Gli1CreERT;Ai14 mice were injected with tamoxifen at P5 and P7


title: TS36

description: results.act.female.csv.gz

tag: gender

value: low

tag: srbmi

tag: walk score


title: CleftProband_503

value: cleft lip only

value: bilateral


title: CleftProband_512

value: female

value: left


title: TS39

value: buccal cells

tag: bmi category

tag: mvpawk

tag: twin pair number


title: TS34


title: CleftProband_502

tag: Sex

tag: syndromic versus non-syndromic


title: CleftProband_501

value: right


title: TS38


title: TS33


title: Enthesis Gli1 P21

tag: strain

value: post natal day 21


title: E14.5_1

value: embryonic tissue

tag: genotype

value: CD1 IGS wildtype

tag: library name


title: CleftProband_500

value: syndromic

tag: cleft type

tag: cleft laterality

value: unaffected


title: CleftProband_499

value: cleft lip and cleft palate

value: male


title: E13.5_1

tag: cell type


title: TS32


title: CleftProband_498

value: non-syndromic


title: CleftProband_504


title: TS35

description: results.act.female.csv.gz

tag: activity category

value: high


title: CleftProband_497


title: CleftProband_495


title: CleftProband_496


title: Enthesis Gli1 P28

tag: tissue

value: Tendon enthesis

tag: age

value: post natal day 28

value: Gli1CreERT;Ai14 mice were injected with tamoxifen at P5 and P7


title: CleftProband_494

value: bilateral


title: TS31

tag: gender

value: female


title: Enthesis Gli1 P7

value: post natal day 7

tag: treatment


title: TS30

description: results.act.female.csv.gz

value: buccal cells

value: low

tag: srbmi

tag: walk score


title: Enthesis Gli1 P18

value: post natal day 18


title: CleftProband_493

value: cleft palate only

value: not applicable


title: Enthesis Gli1 P11

value: post natal day 11


title: Enthesis Gli1 P14

value: post natal day 14


title: CleftProband_492

value: cleft lip only

value: left

value: unaffected


title: TS29

description: results.act.female.csv.gz

value: high

tag: mvpawk


title: CleftProband_491

value: male

value: non-syndromic

value: cleft lip and cleft palate


title: CleftProband_490


title: Hamp2

city: Dallas

first: Tao

last: Wang

state: Texas

address: 5323 Harry Hines Blvd.

institute: University of Texas Southwestern Medical Center

data_processing:
10X scRNA-seq data was preprocessed using the Cellranger v3.0.2 software
the mkfastq and count commands to process the 10x single-cell RNA-seq output into cells by gene expression count matrix, and used the aggr command to pool sequencing runs from 4 mice. All parameters were set to default except for the “force-cells” parameter, which was set to 4000.
Genome_build: mm10
Supplementary_files_format_and_content: The .h5 files are expression matrix with cell barcodes and gene symbols.

source_name: liver

extract_protocol: Primary hepatocytes were isolated with a two-step collagenase perfusion method described previously. Cells were washed and resuspended in hepatocyte washing medium (Life, 17704024). Single cell libraries were prepared using the 10x Genomics Chromium Single Cell 3’ Reagents Kit v3. For each sample, 8,000 hepatocytes were loaded to target ~5,000 cells after recovery according to the manufacturer's protocol. Single cell libraries were generated and sequenced using the 150 bp paired-end Illumina NextSeq500 in the UTSW Children’s Research Institute Sequencing Facility.

treatment_protocol: four female mice were given Tamoxifen at 6-10 weeks of age to label subsets of hepatocytes with Tomato. Two weeks later, livers were perfused and digested to isolate single cells that underwent single cell RNA-sequencing. No other experimental manipulations were performed.

value: Hamp2-CreERT2; Rosa-LSL-tdTomato

value: liver


title: TS28

value: female

tag: twin pair number


title: CleftProband_489

tag: Sex

value: bilateral


title: CleftProband_488

tag: syndromic versus non-syndromic


title: CleftProband_487


title: Gls2Oat


title: TS27

value: buccal cells


title: Gls2

value: Gls2-CreERT2; Rosa-LSL-tdTomato


title: CleftProband_486


title: TS26

description: results.bmi.female.csv.gz

tag: bmi category


title: CleftProband_484


title: CleftProband_485


title: CleftProband_482


title: Cyp1a2

tag: genotype

value: Cyp1a2-CreERT2; Rosa-LSL-tdTomato


title: CleftProband_483


title: CleftProband_481


title: TS25

description: results.bmi.female.csv.gz

value: low


title: TS24

description: results.act.female.csv.gz


title: CleftProband_479

value: right


title: RORaRORcKO 2

first: Eunha

last: Kim

state: MA

address: 77 Ave Louis Pasteur

department: Immunology

institute: Harvard Medical School

data_processing:
The basecall files were demultiplexed through the Harvard BPF Genomics Core’s pipeline, and the resulting fastq files
Quantification of gene expression was done by a lightweight alignment tool, Salmon (Patro, Rob, et al. “Salmon provides fast and bias-aware quantification of transcript expression.” Nature Methods (2017)) with kmer 31
Genome_build: GRCm38
Supplementary_files_format_and_content: Salmon quantification files (Patro, Rob, et al. “Salmon provides fast and bias-aware quantification of transcript expression.” Nature Methods (2017))

source_name: In vitro differenciated Th17

growth_protocol: 200,000 FACS-sorted naïve CD4 T cells (CD4+ CD8a- CD25- CD44low CD62Lhigh) from C57BL/6J mice were seeded on multiwell plates pre-coated with hamster anti-rat IgG. T cells then activated and expanded by soluble anti-CD3e (145-2C11, 0.25 μg/ml) and anti-CD28(37.51, 1 μg/ml) in Th17 polarizing condition (10 ng/ml mouse IL-6, 0.3 ng/ml human TGFβ1) in RPMI media supplemented with 55 mM β-mercaptoethanol, 1xGlutaMax and 10% fetal bovine serum for 3 days.

extract_protocol:
Qiagen RNeasy Plus Mini Kit according to the manufacture’s protocol
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: 3-OxoLCA, isoLCA or DMSO (vehicle) was added to culture 18 hours after TCR activation at 10 μM final concentration.

value: In vitro differenciated Th17

tag: strain

value: RORaRORcKO

tag: tissue origin

value: Spleen and lymphnoids originated naïve CD4 T cells


title: TS23

description: results.act.female.csv.gz


title: CleftProband_480

tag: cleft type

value: cleft lip only

tag: cleft laterality

value: unaffected


title: RORaRORcKO 1

tag: cell type


title: TS22


title: WT 3-oxoLCA 3

value: WT

tag: agent


title: WT 3-oxoLCA 2


title: CleftProband_478

value: non-syndromic

value: male


title: CleftProband_477

value: cleft lip and cleft palate


title: CleftProband_476

value: cleft palate only

value: not applicable


title: WT 3-oxoLCA 1


title: CleftProband_475

value: female

value: bilateral


title: TS21

value: buccal cells


title: CleftProband_474


title: TS20

tag: walkability category

value: high


title: CleftProband_472


title: WT IsoLCA 3

value: IsoLCA


title: TS19

tag: gender

value: low


title: CleftProband_473


title: WT IsoLCA 2

value: In vitro differenciated Th17


title: CleftProband_471

value: unknown


title: CleftProband_470

value: right


title: CleftProband_469


title: WT IsoLCA 1

value: Spleen and lymphnoids originated naïve CD4 T cells


title: TS18

description: results.act.female.csv.gz

tag: activity category


title: CleftProband_467

value: syndromic

value: unaffected


title: WT DMSO3

value: WT

value: DMSO


title: WT DMSO2


title: CleftProband_466

value: male

value: non-syndromic

value: cleft lip and cleft palate

value: left


title: TS15

value: female

tag: srbmi


title: CleftProband_463


title: WT DMSO1


title: TS14

value: buccal cells

tag: walk score


title: CleftProband_461

value: cleft lip only


title: CleftProband_464


title: CleftProband_462


title: CleftProband_460

value: bilateral


title: CleftProband_459


title: TS13

tag: mvpawk


title: CleftProband_458


title: TS12

tag: twin pair number


title: CleftProband_468

tag: Sex


title: CleftProband_456

tag: syndromic versus non-syndromic


title: Balb-C female mouse liver_S4_L002_Nafion BP2-6.0 [GRC362-24_S4]

city: Research Triangle Park

first: Christopher

last: Corton

state: NC

address: 109 TW Alexander Dr.

institute: US-EPA

data_processing:
Sequencing and base calling were completed using the Illumina NextSeq 500 according to standard methods. The sequenced mRNA reads were mapped to the mouse genome in a Partek Flow suite. Pre and post  alignment QC checks were done on each sample to relate all data for the single sample to the corresponding sequenced reads. The processed data matrix is derived from the Partek output file.
genome build: GRCm38.p5; GCF_000001635.25
Supplementary_files_format_and_content: The processed data matrix contains a single data point per sample that represents the summary of results from the four (4) fastq files produced per sample. Therefore, the sample names (20 total) in the matrix file are the ‘root name’ of the four (4) related metadata sample names (80 total), e.g., data matrix sample GRC362-01_S14 is a summary of data from samples GRC362-01_S14_L001, GRC362-01_S14_L002, GRC362-01_S14_L003 and GRC362-01_S14_L004.

source_name: Balb-C female mouse liver_Nafion BP2-6.0

extract_protocol: The livers tissues (~50-100 mg) were preserved in RNAlater (Qiagen, Valencia, CA) and stored at -80°C until RNA extraction. Total RNA was extracted from tissue samples 2-6 months later with Trizol by polytron homogenization. The resultant RNA was further purified using the RNeasy Mini Kit (Qiagen), and RNA integrity was checked with Bioanalyzer RNAnanochip. Samples were submitted to EPA core facility for processing and analysis using the Illumina mouse whole genome platform and NexSeq 500 system.

treatment_protocol: Balb-C female mice at 10-12 weeks of age were dosed by gavage for 7 consecutive days using 20-gauge stainless steel feeding needles. Doses of 0, 0.03, 0.3, 3.0, and 6.0 mg/kg-day were administered once daily in the afternoon. Control animals were administered an equal volume of deionized water. Four biological replicates consisting of individual animals were included in each dose group.

value: Balb-C

tag: age

value: 10-12 weeks

tag: treatment

value: Nafion BP2

tag: dose

value: 6.0 mg/kg

tag: tissue

value: liver


title: CleftProband_457

tag: cleft type


title: TS17

description: results.act.female.csv.gz

value: high


title: Balb-C female mouse liver_S4_L003_Nafion BP2-6.0 [GRC362-24_S4]

tag: strain


title: Balb-C female mouse liver_S4_L004_Nafion BP2-6.0 [GRC362-24_S4]


title: TS11

tag: cell type


title: CleftProband_454

tag: cleft laterality

value: unaffected


title: CleftProband_455

value: right


title: Balb-C female mouse liver_S4_L001_Nafion BP2-6.0 [GRC362-24_S4]


title: CleftProband_453

value: syndromic


title: TS16


title: CleftProband_465

value: male


title: CleftProband_452

value: non-syndromic


title: Balb-C female mouse liver_S1_L004_Nafion BP2-6.0 [GRC362-23_S1]


title: CleftProband_451

value: cleft lip and cleft palate


title: Balb-C female mouse liver_S1_L003_Nafion BP2-6.0 [GRC362-23_S1]


title: CleftProband_450

value: left


title: CleftProband_449

value: female


title: Balb-C female mouse liver_S1_L002_Nafion BP2-6.0 [GRC362-23_S1]


title: TS9

description: results.walk.male.csv.gz

value: buccal cells

tag: walkability category

value: low


title: CleftProband_448

value: unknown


title: Balb-C female mouse liver_S1_L001_Nafion BP2-6.0 [GRC362-23_S1]

value: Balb-C


title: CleftProband_447


title: CleftProband_446

value: cleft lip only


title: TS7

description: results.act.male.csv.gz

tag: gender


title: TS6


title: Balb-C female mouse liver_S7_L004_Nafion BP2-6.0 [GRC362-22_S7]

value: 10-12 weeks

value: Nafion BP2


title: CleftProband_443


title: CleftProband_444


title: Balb-C female mouse liver_S7_L003_Nafion BP2-6.0 [GRC362-22_S7]

value: 6.0 mg/kg

value: liver


title: TS5


title: CleftProband_445

value: unaffected


title: CleftProband_442


title: TS10

description: results.walk.male.csv.gz

value: high


title: Balb-C female mouse liver_S7_L002_Nafion BP2-6.0 [GRC362-22_S7]


title: CleftProband_439


title: Balb-C female mouse liver_S7_L001_Nafion BP2-6.0 [GRC362-22_S7]


title: CleftProband_440


title: TS3


title: CleftProband_438


title: TS8

description: results.act.male.csv.gz

tag: activity category


title: Balb-C female mouse liver_S10_L004_Nafion BP2-6.0 [GRC362-21_S10]


title: TS2

tag: bmi category

tag: srbmi


title: CleftProband_437


title: CleftProband_435


title: TS1

tag: walk score

tag: mvpawk


title: Balb-C female mouse liver_S10_L003_Nafion BP2-6.0 [GRC362-21_S10]

tag: Sex


title: CleftProband_434

tag: syndromic versus non-syndromic

value: cleft palate only

value: not applicable


title: C2 sogenic clone, BRMS1v2 A273V/A273V, replicate 2

first: Yuan

last: Liu

address: 1275 York Avenue

institute: Memorial Sloan Kettering Cancer Center

data_processing:
CutAdapt version cutadapt-1.6 was used to trim and filter reads. Adapters were trimmed with minimum overlap of 10 bases and reads of less than half original read length were filtered out.
STAR version STAR-STAR_2.5.0a was run using the 2-pass method. First pass was run with the following parameters: --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMattributes All --outSAMunmapped Within. Genome database was then generated using the minimum read length of half desired read length and second pass was run against the new genome file with the same parameters as pass 1.
Expression count matrix was computed using HTSeq version HTSeq-0.5.3. Parameters set were: -m intersection-strict -s no -t exon
Assembly: GRC37/hg19
Supplementary files format and content: tab-delimited file containing raw counts

growth_protocol: RPMI + 10% FBS

extract_protocol:
Total RNA was isolated using the RNeasy mini kit (74104, Qiagen), in accordance with the manufacturer’s protocol
library preparation and RNA sequencing were performed by the Integrated Genomics Operation at MSK using Illumina HiSeq with 50 paired-end reads, and approximately 30 million reads were generated for each sample

tag: cell line

value: epithelial cells

value: BRMS1v2 A273V/A273V


title: CleftProband_433

value: male

value: non-syndromic

value: cleft lip and cleft palate

value: right


title: Balb-C female mouse liver_S10_L002_Nafion BP2-6.0 [GRC362-21_S10]

tag: age

value: female

tag: treatment


title: CleftProband_432


title: C2 isogenic clone, BRMS1v2 A273V/A273V, replicate 1

tag: tissue


title: TS4

value: buccal cells

tag: twin pair number


title: CleftProband_441

tag: cleft type

value: left


title: CleftProband_431

value: syndromic


title: Balb-C female mouse liver_S10_L001_Nafion BP2-6.0 [GRC362-21_S10]

tag: strain

value: Balb-C

value: 10-12 weeks

value: Nafion BP2

tag: dose


title: CleftProband_430

value: cleft lip only

tag: cleft laterality

value: unaffected


title: CleftProband_428


title: C1 isogenic clone, BRMS1v2 A273V/A273V, replicate 2

tag: cell type


title: CleftProband_429

value: bilateral


title: C1 isogenic clone, BRMS1v2 A273V/A273V, replicate 1


title: Balb-C female mouse liver_S2_L004_Nafion BP2-3.0 [GRC362-20_S2]

source_name: Balb-C female mouse liver_Nafion BP2-3.0

value: 3.0 mg/kg

value: liver


title: CleftProband_426


title: CleftProband_425


title: Balb-C female mouse liver_S2_L003_Nafion BP2-3.0 [GRC362-20_S2]


title: Balb-C female mouse liver_S2_L002_Nafion BP2-3.0 [GRC362-20_S2]


title: CleftProband_436


title: CleftProband_424


title: Parental cell lines, BRMS1 WT, replicate 2

value: epithelial cells

value: BRMS1 WT


title: CleftProband_427

value: non-syndromic

value: male


title: Balb-C female mouse liver_S2_L001_Nafion BP2-3.0 [GRC362-20_S2]


title: CleftProband_423

value: cleft lip and cleft palate


title: HeLa cells, BETF enriched cytoplasmic RNA

city: Beijing

first: Haofan

last: Sun

address: Grindtorpsvägen 11 7tr Lgh 1803, Grindtorpsvägen 11, 183 32 Täby

institute: National Center for Protein Sciences, Beijing

data_processing:
To estimate the content of RNA biotypes in libraries, reads were aligned to the hg38 assembly using STAR.
Counting was performed with HTSeq-count using the gene set annotated by GENCODE Release 21 (GRCh38) and using the GTF feature ‘gene’ for counting.
Assembly: hg38
Supplementary files format and content: Tab-separated values files

molecule: cytoplasmic RNA

source_name: Hela

extract_protocol:
RNAs were purified by TRIzol following the manufacturer’s instructions
The RNA samples were prepared with the TruSeq RNA Library Prep Kit v2.

value: Hela

value: Human cervix carcinoma

value: BETF enrichment


title: Balb-C female mouse liver_S8_L004_Nafion BP2-3.0 [GRC362-19_S8]

value: female


title: 9M, ovoid cells

city: Moscow

first: Artem

last: Grigorov

address: Ulitsa Miklukho-Maklaya, 16/10

department: Genomics and Postgenomic Technologies

country: Russia

institute: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry

data_processing:
Sequenced reads were mapped to M.smegmatis MC2 155 genome using bowtie2 with parameters -p 4 (and --local --dovetail in case paired-end sample)
Reads counting was performed using FeatureCounts (Annotation of the genome was retrieved from Mycobrowser). For every annotated gene, the number of reads (or fragments) overlapping the gene at least by one nucleotide was calculated. Only unambiguously mapped reads were used in a subsequent analysis. The files "Counts_XX.txt" represent these counts (File "Counts_all.txt" contains values for all samples).
Assembly: NC_008596.1

organism: Mycolicibacterium smegmatis MC2 155

source_name: bacterial culture

growth_protocol: Cells were first grown in Nutrient broth (“Himedia”, India) in the presence of 0.05 % Tween-80 at 37 °C and 200 rpm for 24 h. The resulting culture was added at a concentration of 105–106 cells/ml to 250 ml of Sauton (pH 7.0, for “active” cultures) or modified Sauton (pH 6.0-6.2, for “dormant” cultures) medium. The modified Sauton medium contained (per litre): 0.5 g KH2PO4; 1.4 g MgSO4·7H2O; 4 g L-asparagine; 60 ml glycerol; 0.05 g ferric ammonium citrate; 2 g sodium citrate, and 0.1 ml 1% ZnSO4·7H2O, supplemented with 0.05% Tween-80. The modification included a decrease of the initial pH to 6.0–6.2 (no titration with NaOH) (Kudykina et al., 2011). Cultures were incubated at 37°C under constant shaking for 10–20 days, and pH values were periodically measured. Active control cultures were harvested at late exponential/pre-stationary phase. Dormant cells were harvested when cultures entered the post-stationary phase, changing the color and reaching pH 6.0–6.2 (after 13–15 days of cultivation). Cultures were transferred to plastic-capped tubes (50 ml) and maintained under static conditions without agitation at room temperature for up to 30 days post inoculation.

extract_protocol:
Active and dormant (ovoid) cultures were harvested by centrifugation at 7000 rpm for 15 min and washed 3 times with an ice-cold PBSm buffer containing (per liter) 8 g NaCl, 0.2 g MgCl2, and 0.24 g Na2HPO4 (pH 7.0). The bacterial pellet was re-suspended in the same buffer containing phenylmethanesulfonyl fluoride (PMSF) then lysed by using zirconium beads on a bead beater homogenizer (MP Biomedicals FastPrep-24) for 30 sec, three times. The bacterial lysate was centrifuged at 13000 rpm for 10 min at 4 °C. The supernatant was separated into membrane and cytosolic fractions using ultracentrifugation at 105000g and 4 °C for 2 h. The membrane fraction was washed with PBSm using ultracentrifugation. For RNA isolation the pellet was re-suspended in ice-cold 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH 8.0) containing 5 mM EDTA and the zwitterionic detergent CHAPS (3-(3-cholamidopropyl) dimethylammonio-1-propanesulfonate hydrate (2%) (w/v)).
Total RNA waw isolated by phenol-chloroform extraction as described previously [Rustad TR, Roberts DM, Liao RP, Sherman DR. Isolation of mycobacterial RNA. Methods Mol Biol. 2009;465:13–21.]
After isolation RNA was treated with Turbo DNase (Life Technologies, USA) to remove traces of genomic DNA. Amounts and purity of RNA were determined spectrophotometrically; integrity of RNA was assessed in 1% agarose gel. Sequencing libraries were generated using the resulting RNA and NEBNext Ultra II Directional RNA Library Prep Kit (NEB, USA) according to the manufacturers' protocol.

value: bacterial culture

tag: state

value: dormant (ovoid)