title: Case 57: Aneurysmal bone cyst

description: Aneurysmal bone cyst


title: Run 1 Peripheral Immune cells

status: Public on Oct 18 2023

type: SRA

city: Frankfurt

first: Julius

middle: C

last: Enssle

address: Theodor-Stern-Kai 7

department: Hematology/Oncology

country: Germany

institute: University Hospital Frankfurt

description: read1 file contains cell barcode and UMI; read2 file contains transcript, Antibody Derived Tag or Sample Tag

library_source: other

data_processing:
After Illumina sequencing, the SevenBridges platform (Seven Bridges Genomics, https://www.sevenbridges.com) was used to upload the FASTQ files and reference sequences for mRNAs and AbSeqs. The data was then processed using the BD Rhapsody analysis pipeline (BD Biosciences) with default settings. In summary, low quality read pairs were ejected before the leftover R1 reads were mapped for the identification of cell label and unique molecular identifier (UMI) sequences. Second, the R2 reads were annotated to the reference sequences allowing reads with identical cell label, UMI sequence and reference gene to be collapsed into a single molecule. With the help of a recursive substitution error correction (RSEC) algorithm, the molecule counts were corrected for sequencing errors. Then, Sample Tags were mapped to cells and cell counts were determined to create RSEC-adjusted molecule count matrices.
Assembly: hg18 reference genome and antibody dependent tags (T_NK_NKT_49AbSeqPanel_ref_file.fasta).
Supplementary files format and content: BD Rhapsody pipeline output files: *_Sample_Tag_Calls_Donors.csv, *_RSEC_MolsPerCell.csv
Library strategy: CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing)

molecule: other

organism: Homo sapiens

source_name: peripheral blood

extract_protocol:
Thawed cells were washed and 1 million cells were stained per donor. As recommended, equal amounts (in µg) of fluorescent-labelled (FACS) and oligonucleotide-labelled (AbSeq) anti-CD3 and anti-CD56 antibodies were added to allow appropriate antibody staining for both read-outs. Both antibodies were from the same clone (SK7 for CD3 and NCAM16.2 for CD56). After 10 minutes of incubation on ice, the remaining fluorescent-labeled antibodies (anti-CD19, anti-CD14, anti-CD45) and sample tags were added and incubated for another 30minutes. Samples were washed and labelled with 7AAD for live/dead discrimination. Cells were sorted with the BD FACS Aria Fusion device (BD Biosciences) with a 70µm nozzle under sterile conditions. Per donor, equal amounts of T-cells (defined as 7AAD-, CD45+, CD19- CD14-, CD56-, CD3+; 3x10E4 cells), B cells (7AAD-, CD45+, CD19+, CD14-; 1x10E4 cells), NK-cells (defined as, 7AAD-, CD45+, CD19-, CD14-, CD56+ CD3-; 1x10E4 cells) and NKT-cells (defined as 7AAD-, CD45+, CD19-, CD14-, CD56+ CD3+; 5x10E3 cells) were aimed and sorted in the same tube to gain constant cell distributions between donors. Afterwards, single cell sequencing was performed using a BD Rhapsody™ Single-cell analysis system (BD Biosciences).
Following the instructions of the BD Rhapsody whole transcriptome analysis (WTA), AbSeq and Sample Tag amplification kit (BD Biosciences, cat# 633801), DNA libraries were generated. The final libraries including indexes for Illumina sequencing were assessed regarding their concentration and size with a Qubit 4 Fluorometer using a High Sensitivity dsDNA kit (ThermoFisher Scientific, cat# Q32851) and an Agilent 4150 TapeStation using a High Sensitivity D1000 ScreenTape (Agilent, cat# 5067-5584, 5067-5585). At last, library pools with a final concentration of 1 nM were mixed and loaded in multiple runs (75 bp paired-end) on a NextSeq2000 sequencer (Illumina) together with 20 % PhiX DNA.

tag: tissue

value: peripheral blood

tag: cell type

value: T-, NK-, NKT-, B-cells

tag: library type

value: mRNA  + ADT + sample barcode

tag: antibodies/tags

value: Sample tags:1-Donor1_TP3, 2-Donor1_TP5, 3-Donor2_TP3, 4-Donor2_TP5, 5-Donor3_TP3, 6-Donor3_TP5, 7-Donor4_TP3, 8-Donor4_TP5, 9-Donor5_TP3, 10-Donor5_TP5, 11-Donor6_TP3, 12-Donor6_TP5


title: Case 71: Aneurysmal bone cyst

type: genomic

city: Heidelberg

first: Daniel

last: Schrimpf

address: Im Neuenheimer Feld 224

department: Neuropathology

institute: University Hospital Heidelberg, Intitute of Pathology

description: Aneurysmal bone cyst

hyb_protocol: Illumina protocol

scan_protocol: Illumina iScan System according to the manufacturer's protocol

data_processing:
Bioconductor (Version 3.3) package minifi (Version 1.18) using function 'preprocessIllumina'  Average beta
Average Beta

label: Cy3 and Cy5

molecule: genomic DNA

source_name: Aneurysmal bone cyst

label_protocol: Illumina protocol

extract_protocol: Illumina protocol

treatment_protocol: An automated extraction was done with a Maxwell system (Promega, Fitchburg, WI, USA) and the Maxwell 16 FFPE Plus LEV DNA Purification Kit according to the manufacturer’s instructions.

tag: material

value: FFPE


title: M3-Right-14-Neuron-RNA-R1

city: Blacksburg

first: Chang

last: Lu

state: VA

address: 235 Goodwin Hall, 635 Prices Fork Road, Virginia Tech

department: Chemical Engineering

country: USA

institute: Virginia Tech

description: MOWChIP-seq data. Name format: Mouse number-Left/right hemisphere-Slice number-Neuron/glia-Marker-Replicate number

library_source: transcriptomic

data_processing:
Raw reads were trimmed with TrimGalore 0.4.1
Trimmed reads were aligned using bowtie 1.1.2. Uniquely mapped reads were filtered for known blacklisted genome regions using Samtools and bedtools to remove ChIP-seq artifacts.
Reads were extended by 100 bp on either ends
Sample was normalized and input subtracted. The reference genome was then divided into 100-bp bins and ChIP-seq signal for each bin was counted for both ChIP and input samples.
Sample was converted to BigWig format
Assembly: mm10
Supplementary files format and content: Normalized ChIP-seq and RNA-seq signal in BigWig format

molecule: total RNA

organism: Mus musculus

source_name: C57BL/6J brain

extract_protocol:
Nuclei extraction from dissected brain tissue was performed using previously described protocol with minor modifications. All steps were conducted on ice and centrifugation was conducted at 4 ℃. A neocortex slice was thawed on ice and transferred to a glass tissue homogenizer (Sigma, d9036-1SET). 3 ml of ice-cold nuclei extraction buffer was added [0.32 M sucrose, 5mM CaCl2, 2 mM Mg(Ac)2 , 0.1 mM EDTA, 10 mM tris-HCl, 0.1% (v/v) Triton X-100, freshly added 1% protease inhibitor cocktail (PIC, Sigma-Aldrich), 0.1 mM PMSF, 1 mM DTT, and 0.06 U/µl ribonuclease (RNase) inhibitor (2313A,Takara Bio, needed when mRNA extraction was performed)]. The tissue was homogenized with 10 times of slow douncing using a loose pestle and 15 times of douncing using a tight pestle (D9063, Sigma-Aldrich). The homogenate was filtered through a 40-µm cell strainer (22363547, Thermo Fisher Scientific) into a 15-ml centrifuge tube and centrifuged at 1000 rcf for 10 min at 4 ℃. Supernatant was discarded and the pellet was resuspended in 0.5 ml of ice-cold nuclei extraction buffer. 0.75 ml of 50% (w/v) iodixanol (made by mixing 4 ml of OptiPrepTM gradient (Sigma-Aldrich) and 0.8 ml of diluent [150 mM KCl, 30 mM MgCl2, and 120 mM tris-HCl]) was added. The suspension was gently mixed by inverting the tube up and down 5 times and then centrifuged at 10,000 rcf for 20 min. Supernatant was carefully removed and the pellet incubated with 100 µl of 2% (w/v) normal goat serum (50062Z, Life Technologies) in Dulbecco’s PBS (DPBS, Life technologies) on ice. The pellet was then resuspended and transferred into a new Eppendorf tube with an addition of 200 µl of 2% normal goat serum. To differentiate neurons from glia, 10 µl of 2 ng/µl Anti-NeuN antibody conjugated with Alexa 488 (MAB377X, EMD Millipore) was mixed with the suspension. The mixture was incubated at 4 ℃ for 1 h on a rotator. The nuclei solution was sorted using FACS (BS FACSAria, BD Bioscience) to separate neuronal (NeuN+) and glial (NeuN-) fractions.   Chromatin fragmentation:  M1-2: 4,000 nuclei per replicate was used so that both neuronal and glial fractions of every neocortex slice sample yielded enough material for two ChIP-seq replicates. The nuclei suspension (containing 8,000 nuclei) was topped up to 1 ml using Dulbecco’s PBS (DPBS; 14190144, Life Technologies). 200 µl of 1.8M sucrose, 10 µl of 1M CaCl2, and 3 µl of 1M Mg(Ac)2 were then added sequentially. The sample was mixed gently by inverting the tube up and down and then incubated for 15 min on ice. The suspension was centrifuged at 3,000 rpm for 15 min at 4 ℃. Supernatant was carefully removed, and the pellet was resuspended in 10 µl DPBS. 0.1 µl of PIC, 0.1 µl of 100 mM PMSF and 10 µl of lysis buffer [4% Triton X-100, 100 mM tris (pH 7.5), 100 mM NaCl, and 30 mM MgCl2] were added, mixed by vortexing and then incubated at room temperature for 10 min. 1 µl of 0.1 M CaCl2 and 0.4 µl of 100 U/µl MNase (88216, Thermo Fisher Scientific) were then added, mixed by vortexing and incubated at room temperature for 10 min. 2.22 µl of 0.5 M EDTA (pH 8) was added to quench the rection. The sample was vortexed and incubated on ice for 10 min before it was centrifuged at 16,100 g for 5min. The supernatant (~20 µl, containing chromatin) was transferred to a new tube and 60 µl of IP buffer [20 mM Tris-HCl, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100] was added to obtain a total volume of ~80 µl. The solution was finally split for two ChIP-seq replicates. The chromatin was stored on ice before loaded into the MOWChIP device.  M4-5: 200 µl of sorted neuronal nuclei suspension from one neocortex slice sample was used to prepare chromatin (containing ~30,000 nuclei). 2 µl of PIC, 2 µl of 100 mM PMSF and 200 µl of lysis buffer were added, mixed by vortexing and incubated at room temperature for 10 min. 4 µl of 1 M CaCl2 and 5 µl of 100 U MNase (88216, Thermo Fisher Scientific) were added, mixed by vortexing and incubated at room temperature for 10 min. 44 µl of 0.5 M EDTA was then added, mixed by vortexing and incubated on ice for 10 min. The mixture was finally centrifuged at 16,100g for 5 min at 4 °C. Supernatant containing fragmented chromatin (~ 440 µl) was collected into a new 1.5 ml tube and placed on ice before use. 70 µl of chromatin (containing chromatin from ~4,000 nuclei) was used for each MOWChIP-seq assay for consistency with data on M1-2.  Antibody coating on IP beads:  5 µl of Dynabeads Protein A (10002D, Thermo Fisher Scientific) were used in each MOWChIP assay. Beads were washed twice with 150 µl of IP buffer [20 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100] and then resuspended in 150 µl of IP buffer. An antibody that targets a specific modified histone was added to coat the beads: anti-H3K27ac (ab4729, Abcam) at 0.125 µg/assay or anti-H3K27me3 (39155, Active Motif) at 0.5 µg/assay. The bead/antibody suspension was incubated on a rotator at 4 °C overnight. Beads were then washed with IP buffer twice and resuspended in 5 µl of IP buffer for use in MOWChIP.  MOWChIP:  The MOWChIP process, including device fabrication and operation, was performed following our published protocol (Zhu, B., Hsieh, YP., Murphy, T.W. et al. MOWChIP-seq for low-input and multiplexed profiling of genome-wide histone modifications. Nat Protoc 14, 3366–3394 (2019).).
ChIP-seq libraries:  ~4,000 neuronal or glial nuclei were used to produce each ChIP-seq library and two technical replicates were generated on each neocortex slice sample. The input libraries were created using the same starting quantity of nuclei (~ 4,000 per library) with the MOWChIP step skipped. Libraries were constructed using 2S Plus DNA Library kit (10009877, IDT) and 2S CDI Adapter S1 indexing kit (10009895, IDT) following the manufacturer’s instructions. 5% (v/v) EvaGreen (31000, Biotium) was added to the amplification mixture to monitor the progress of library amplification. Amplification was stopped once an increase of 2,000 RFU was reached. Library was eluted to 10 µl of low EDTA TE buffer.  mRNA-seq:  mRNA libraries were prepared following Smart-seq2 protocol55 with minor modifications. ~7,500 neuronal nuclei (from M3) were used to generate each RNA-seq library, and each slice sample has two technical replicates. 50 μl of neuronal suspension containing about 7,500 nuclei was used for RNA extraction using RNeasy Mini kit (QIAGEN) and RNase-Free DNase Set (79254, QIAGEN). Extracted RNA was concentrated using ethanol precipitation and resuspended in 5 μl of RNase-free water. 4.6 μl of RNA solution, 2 μl of 10 μM oligo-dT primer and 2 μl of 10 mM dNTP mix were mixed, incubated at 72°C for 3 mins and then stored on ice. 11.4 μl of reverse-transcription mix (made from 1 μl of SuperScript II reverse transcriptase (200 U/μl), 0.5 μL of RNase inhibitor (40 U/μl), 4 μL of Superscript II first-strand buffer, 1 μl of DTT (100mM), 4 μl of 5 M Betaine, 0.12 μl of 1 M MgCl2, 0.2 μl of TSO (10 μM), 0.58 μl of nuclease-free water) was added to the RNA solution and incubated at 42°C for 90 min, followed by 10 cycles of (50°C for 2 min, 42°C for 2 min), and at 70°C for 15 min. 25 μl KAPA HiFi HotStart ReadyMix, 0.5 μl of 10 μM IS PCR primers, 2.5 μl of 20x Evagreen, and 4 μl of RNase-free water were then added. cDNA was amplified by incubation at 98°C for 1 min, followed by 9-11 cycles (to achieve an increase of 50-100 RFU) of (98°C for 15 s, 67°C for 30 s, 72°C for 6 min). After amplification, cDNA was purified using 0.8× SPRIselect beads. Tn5 transposase (14-17 mg/ml) was produced following a published protocol56.  Tn5 transposase and T5/T7 transposons were assembled to form Tn5 transposome using a published protocol57. Briefly, 1.5 μl of 100 μM T5 transposon (IDT) was mixed with 1.5 μl of 100 μM pMENTS (a 5'-phosphorylated 19-bp mosaic end complementary oligonucleotide, IDT), while 1.5 μl of 100 μM T7 transposon (IDT) was mixed with 1.5 μl of 100 μM pMENTS. 3 μl of T5-pMENTS solution and 3 μl of T7-pMENTS solution were separately incubated at 95℃ for 5 min followed by a slow ramp to 25℃ at -0.1℃/sec. 2.5 μl of T5-pMENTS solution was then mixed with 2.5 μl of Tn5 transposase (14-17 mg/ml), while 2.5 μl of T7-pMENTS solution was mixed with 2.5 μl of Tn5 transposase. T5-Tn5 solution and T7-Tn5 solution were separately incubated at 37℃ for 1 h to form T5-Tn5 transposome and T7-Tn5 transposome, respectively. 4 μl of T5-Tn5 transposome and 4 μl of T7-Tn5 transposome were mixed to form 8 μl of assembled Tn5 transposome before it was mixed with 1 μl cDNA (containing ~500 pg cDNA) and 1 μl reaction buffer (TNP92110, Lucigen). The reaction mixture was incubated at 37 ℃ for 1 h. 1 μl of 10× stop solution (TNP92110, Lucigen) was added to the reaction mixture to stop tagmentation. Two rounds of 1× SPRIselect beads cleanup were performed to purify tagmented cDNA. The tagmented cDNA was then eluted in 10 μl low EDTA TE buffer. 25 μl KAPA HiFi HotStart ReadyMix was activated at 98 ℃ for 30 sec and then mixed with 9.5 μl tagmented cDNA and 10 μl Nuclease-free water, followed by incubation at 72 ℃ for 5 min. 1.5 μl of 25 M P5 primer, 1.5 μl of 25 M P7 primer and 2.5 μl 20x Evagreen were then added. The following amplification program was used to achieve a 300-400 RFU increase: 98 ℃ for 30 s; 11-12 cycles of (98 ℃ for 10 s, 63 ℃ for 30 s, 72 ℃ for 30 s) and 72 ℃ for 1 min. Amplified library was purified with 0.8× SPRIselect beads and eluted in 8 μl low EDTA TE buffer.  Library quality control and sequencing:  Library size and concentration were measured by Tapestation (Agilent) and KAPA Library Quantification Kit (07960140001, Roche), respectively. Libraries were pooled for sequencing by Illumina HiSeq 4000 with single-end 50 nt reads or NovaSeq 6000 in S4 200 mode with paired-end 100 nt reads. Averagely, 19 million reads were generated for each ChIP-seq library, 12 million reads for each ChIP-seq input library, and 16 million reads for each RNA-seq library.
ChIP-seq, RNA-seq

treatment_protocol: All procedures were conducted in accordance with NIH guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at Virginia Tech. Adult (P56) C57BL/6J male mice were purchased from the Jackson Laboratory and housed on a 12 h light/dark cycle at 23 °C with food and water ad libitum for 2 weeks before experiments.  M1-M3 were euthanized using compressed CO2 followed by cervical dislocation. Mouse brain was then isolated and cut into coronal slices with a thickness of 0.5 mm using an adult mouse brain slicer (BSMAS005-1, Zivic Instruments) on ice. Left and right neocortex sections were dissected from each slice and immediately frozen in dry ice. The dissected neocortex slice samples (a total of 18-21 slices for either left or right hemisphere of a neocortex) were stored at -80 ℃ until use.M4 and M5 were treated for the study of chemically induced seizure. 2 mg/ml kainic acid (K0133, LKT Laboratories) in sterile saline was prepared freshly. M4 was injected intraperitoneally with repeated 5 mg/kg doses of kainic acid at 30 min intervals until behavioral seizures were observed (after 2 doses). M5 (control) was injected with the same amount of sterile saline at 30 min intervals for 2 doses. M4 and M5 were euthanized by cervical dislocation and death was confirmed by bilateral thoracotomy. Mouse brain was processed to extract 0.5 mm-thick left and right coronal neocortex slices and the slice samples were stored at -80 °C until use.

value: brain

tag: strain


title: M3-Right-13-Neuron-RNA-R2


title: Case 55: Peripheral giant cell granuloma of the jaws


title: K562_library2_2_gRNA amplification

city: Beijing

first: Yue

last: Huo

state: Beijing

address: Dongcheng District, Dongdan 3 Tiao 5 Hao

department: State Key Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences

country: China

institute: Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College

description:
gRNA amplification library for 10X cDNA samples (K562_library2_2)
library2_gRNA_whitelist.txt.gz
Sample 13

data_processing:
Library strategy: amplified gRNA sequencing
All 10x scRNA-seq datasets were processed with the Cell Ranger (v3.0.2) pipeline (https://www.10xgenomics.com/) and were mapped to the Homo sapiens genome (Ensembl GRCh38.p12). This step yields a total of 71,014 cells, and an average of 52,758 reads per cell; 3,326 median genes per cell, and a median of 17,478 UMIs per cells.
Libraries of gRNA amplification were separately indexed and sequenced as spike-ins alongside the scRNA-seq libraries. UMI and cell-barcode assignments were made for each read by processing these samples with the Cell Ranger (v3.0.2) 'count' tools. Then, we combined the output bam files and the whitelist of guides sequence in our libraries to obtain the cell-barcode and guide pairwise using 'get_barcodes.py' (Hill et al., 2018) with parameters: "--search_seq TTGTGGAAAGGACGAAACACCG --all_reads --barcode_length 20 --no_swalign --chimeric_threshold 0.2". The similar chimeric sequences and reads unassigned to a gRNA were removed. Cell barcodes detected in the gRNA amplification libraries were then matched with cell-barcodes detected in the regular 10x scRNA-seq libraries. We take all whitelist sequences that have at least one UMI counts and account for over 7.5% of the whitelist reads assigned to a given cell using 'preprocess_cfg.R' tools (Hill et al., 2018).
Genome_build: Ensembl GRCh38.p12
Supplementary_files_format_and_content: Barcodes detected, genes detected and matrix h5 files are provided for each sample
Supplementary_files_format_and_content: *whitelist.txt: gRNA sequence list

organism: synthetic construct

source_name: K562 cell line

growth_protocol: (Sample 8-14) Human erythroleukemia cell line K562 was maintained in RPMI1640 supplemented with 10% FBS (Gibco, Carlsbad, CA, USA).

extract_protocol: The enriched PCR procedure was performed as described previously(Hill et al., 2018) with the following modifications. A heminested PCR starting from 40 ng of full-length cDNA was used to enrich for gRNA barcodes. Q5 High-Fidelity DNA Polymerase (New England BioLabs) was used for PCR amplification according to the following PCR protocol: (1) 98°C for 45 s, (2) 98°C for 15 s, 65°C for 20 s, then 72°C for 60 s (14-16 cycles). PCRs were cleaned with Zymo DNA Clean & Concentrator-5 columns (Zymo, Orange County, CA, USA) and 15 µl of a 1:2 dilution of the first PCR and 1:2 dilution of the second PCR were carried in each reaction. Then fragments of length 400-500 bp were selected using 8% TBE gel.

treatment_protocol: gRNAs were connected to the tandem double gRNA expression plasmid (pLV-hEF1a-EGFP-2A-Puro-U6-gRNA1-7sK-gRNA2) to obtain the gRNA libraries expressing EGFP green fluorescence. 3×106 of K562 cells in 7.5 ml media plus 8 µg/ml polybrene were transduced, with either of the gRNA-EGFP (MOI ~4) or Cas9-mCherry (MOI ~50) expressing viruses that had been packaged singly. At 72 hours post-infection, gRNA-EGFP+/Cas9-mCherry+ cells were collected by flow-cytometry and subjected to Single-cell RNA-seq.

tag: treatment

value: tranduced with a CRISPR KO library 2(molncRNA(-/-)+IC gRNA)

tag: cell line


title: Case 53: Peripheral giant cell granuloma of the jaws


title: K562_library2_1_gRNA amplification

description:
gRNA amplification library for 10X cDNA samples (K562_library2_1)
library2_gRNA_whitelist.txt.gz
Sample 12


title: Case 52: Peripheral giant cell granuloma of the jaws


title: M3-Right-13-Neuron-RNA-R1


title: Case 51: Peripheral giant cell granuloma of the jaws


title: M3-Right-15-Neuron-RNA-R1


title: M3-Right-12-Neuron-RNA-R2


title: K562_library2_3_gRNA amplification

description:
gRNA amplification library for 10X cDNA samples (K562_library2_3)
library2_gRNA_whitelist.txt.gz
Sample 14


title: Case 50: Peripheral giant cell granuloma of the jaws


title: Case 48: Peripheral giant cell granuloma of the jaws


title: K562_library1_3_gRNA amplification

description:
gRNA amplification library for 10X cDNA samples (K562_library1_3)
library1_gRNA_whitelist.txt.gz
Sample 10

value: tranduced with a CRISPR KO library 1(molncRNA(-)+IC gRNA)


title: Case 46: Peripheral giant cell granuloma of the jaws


title: Case 47: Peripheral giant cell granuloma of the jaws


title: Case 49: Peripheral giant cell granuloma of the jaws


title: K562_library1_2_gRNA amplification

description:
gRNA amplification library for 10X cDNA samples (K562_library1_2)
library1_gRNA_whitelist.txt.gz
Sample 9


title: Case 54: Peripheral giant cell granuloma of the jaws


title: Case 43: Non-ossifying fibroma

description: Non-ossifying fibroma

source_name: Non-ossifying fibroma


title: M3-Right-11-Neuron-RNA-R2


title: Case 44: Non-ossifying fibroma

description: Non-ossifying fibroma


title: M3-Right-12-Neuron-RNA-R1


title: M3-Right-10-Neuron-RNA-R2


title: Case 42: Non-ossifying fibroma

description: Non-ossifying fibroma


title: Case 41: Non-ossifying fibroma

description: Non-ossifying fibroma


title: K562_library3_1_gRNA amplification

description:
gRNA amplification library for 10X cDNA samples (K562_library3)
library3_gRNA_whitelist.txt.gz
Sample 11

value: tranduced with a CRISPR KO library 3(NTC gRNA)


title: K562_library1_1_gRNA amplification

description:
gRNA amplification library for 10X cDNA samples (K562_library1_1)
library1_gRNA_whitelist.txt.gz
Sample 8


title: Case 40: Non-ossifying fibroma

description: Non-ossifying fibroma


title: Drosophila_stage5-10_fixed_wild-type#3

city: Pasadena

first: Angelike

last: Stathopoulos

state: CA

address: 1200 E Calif Blvd

institute: Caltech

description: 3' mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript

library_source: transcriptomic single cell

data_processing:
Single-cell RNA-seq data were processed using 10X Cellranger 7 software. Customized dm6-transgene reference annotations were loaded to the pipeline for read mapping and gene quantification.
Assembly: dm6 with mCD8-GFP transgene
Supplementary files format and content: Tab separated txt and tsv table files and matrix files

organism: Drosophila melanogaster

source_name: embryo

extract_protocol:
manual homogenization with Douncer, 5min Accumax treatment, wash, 30µm filter, magnetic beads-based dead cell removal, methanol fixation for sample#3
standard protocol as recommended by 10x Genomics
single cell RNA-seq library preparation was performed with 10x Chromium v3

value: embryo

tag: genotype


title: M3-Right-10-Neuron-RNA-R1


title: Drosophila_stage5-10_live_wild-type#2


title: Case 38: Non-ossifying fibroma

description: Non-ossifying fibroma


title: Case 37: Non-ossifying fibroma

description: Non-ossifying fibroma


title: molnc301b_LacZ

description: ChIRP-seq of molnc-301b in K562 cell line

library_source: genomic

data_processing:
(Sample 60-61) For ChIRP-seq: Reads were aligned to Homo sapiens genome using Bowtie2. Only reads with mapping quality score >30 were retained for further analysis by SAMtools. And per-base coverage was normalized in accordance with previous studies. For each base pair of the genome, true coverage of this base in this sample was defined as the minimum coverage of the even lane and odd lane and a SAM file was generated for peak calling. Peaks of each sample were then called using MACS2 against its corresponding input.
Assembly: Ensembl GRCh38.p5
Supplementary files format and content: peak text file
Library strategy: ChIRP-seq

growth_protocol: (Sample 60-61) For ChIRP-seq: Human erythroleukemia cell line K562 was maintained in RPMI1640 supplemented with 10% FBS (Gibco, Carlsbad, CA, USA).

extract_protocol:
About 2×107 K562 cells were crosslinked by 1% glutaraldehyde followed by sonication. Lysates were clarified from sonicated chromatin and RNA-DNA complexes were isolated by antisense biotinylated oligos on streptavidin beads according to the protocol published by Chu et al., 2011. Then the DNA was purified for subsquential processes.
Libraries were prepared according to NEB's instructions accompanying the NEBNext Ultra II DNA Library Prep Kit (E7103S) and NEBNext Multiplex Oligos for Illumina (E7335S).

tag: lncrna probes

value: LacZ

value: K562 cell line

tag: sequencing type

value: ChIRP-seq


title: Case 45: Non-ossifying fibroma

description: Non-ossifying fibroma


title: M3-Right-09-Neuron-RNA-R2


title: M3-Right-11-Neuron-RNA-R1


title: M3-Right-09-Neuron-RNA-R1


title: M3-Right-08-Neuron-RNA-R2


title: Case 26: Chondroblastoma

description: Chondroblastoma

source_name: Chondroblastoma


title: Case 24: Chondroblastoma

description: Chondroblastoma


title: Case 23: Chondroblastoma

description: Chondroblastoma


title: K562_library2_3

description:
scRNA-seq with molncRNA(-/-) and internal control gRNAs, 10X lane 3
Sample 7

data_processing:
All 10x scRNA-seq datasets were processed with the Cell Ranger (v3.0.2) pipeline (https://www.10xgenomics.com/) and were mapped to the Homo sapiens genome (Ensembl GRCh38.p12). This step yields a total of 71,014 cells, and an average of 52,758 reads per cell; 3,326 median genes per cell, and a median of 17,478 UMIs per cells.
Libraries of gRNA amplification were separately indexed and sequenced as spike-ins alongside the scRNA-seq libraries. UMI and cell-barcode assignments were made for each read by processing these samples with the Cell Ranger (v3.0.2) 'count' tools. Then, we combined the output bam files and the whitelist of guides sequence in our libraries to obtain the cell-barcode and guide pairwise using 'get_barcodes.py' (Hill et al., 2018) with parameters: "--search_seq TTGTGGAAAGGACGAAACACCG --all_reads --barcode_length 20 --no_swalign --chimeric_threshold 0.2". The similar chimeric sequences and reads unassigned to a gRNA were removed. Cell barcodes detected in the gRNA amplification libraries were then matched with cell-barcodes detected in the regular 10x scRNA-seq libraries. We take all whitelist sequences that have at least one UMI counts and account for over 7.5% of the whitelist reads assigned to a given cell using 'preprocess_cfg.R' tools (Hill et al., 2018).
Genome_build: Ensembl GRCh38.p12
Supplementary_files_format_and_content: Barcodes detected, genes detected and matrix h5 files are provided for each sample
Supplementary_files_format_and_content: *whitelist.txt: gRNA sequence list

growth_protocol: (Sample 1-7) Human erythroleukemia cell line K562 was maintained in RPMI1640 supplemented with 10% FBS (Gibco, Carlsbad, CA, USA).

extract_protocol: The libraries were prepared using a Single Cell 3' Library Gel Bead kit V2 (10x Genomics, 120237) following the manufacturer's instructions.


title: K562_library2_2

description:
scRNA-seq with molncRNA(-/-) and internal control gRNAs, 10X lane 2
Sample 6


title: Case 22: Chondroblastoma

description: Chondroblastoma


title: Case 25: Chondroblastoma

description: Chondroblastoma


title: Case 21: Chondroblastoma

description: Chondroblastoma


title: M3-Right-08-Neuron-RNA-R1


title: M3-Right-07-Neuron-RNA-R2


title: Case 20: Chondroblastoma

description: Chondroblastoma


title: K562_library3

description:
scRNA-seq with non-targeting control gRNAs, 10X lane 1
Sample 4


title: K562_library2_1

description:
scRNA-seq with molncRNA(-/-) and internal control gRNAs, 10X lane 1
Sample 5


title: Case 19: Chondroblastoma

description: Chondroblastoma


title: Case 39: Non-ossifying fibroma

description: Non-ossifying fibroma


title: Case 18: Chondroblastoma

description: Chondroblastoma


title: molnc301b_Input_LacZ


title: M3-Right-06-Neuron-RNA-R2


title: M3-Right-07-Neuron-RNA-R1


title: K562_library1_3

description:
scRNA-seq with molncRNA(-) and internal control gRNAs, 10X lane 3
Sample 3


title: Case 36: Non-ossifying fibroma

description: Non-ossifying fibroma


title: Case 17: Chondroblastoma

description: Chondroblastoma


title: Case 15: Cherubism

description: Cherubism

source_name: Cherubism


title: M3-Right-06-Neuron-RNA-R1


title: Case 14: Cherubism

description: Cherubism


title: M3-Right-05-Neuron-RNA-R2


title: K562_library1_1

description:
scRNA-seq with molncRNA(-) and internal control gRNAs, 10X lane 1
Sample 1


title: Case 13: Cherubism

description: Cherubism


title: Case 10: Central giant cell granuloma of the jaws


title: Case 11: Cherubism

description: Cherubism


title: Case 8: Central giant cell granuloma of the jaws


title: Case 7: Central giant cell granuloma of the jaws


title: M3-Right-04-Neuron-RNA-R2


title: RPF_molnc301b_m2_rep2

description: Ribo-seq after molnc301b_m2 overexpression

data_processing:
Library strategy: Ribo-seq
For Ribo-seq and RNA-seq: Raw reads were trimmed by Cutadapt. Then, reads with distinct inline barcodes were demultiplexed using in-house scripts, and the 10-mer random sequence was appended to the reads name in bam file for later usage. Low quality reads were filtered by trimmomatic with parameter "SLIDINGWINDOW:4:15". In Ribo-seq library, reads length in 25-50 bp were retained for subsequent analysis. rRNA reads were removed by aligning reads with rRNA reference sequence on NCBI database by bowtie. Cleaned reads were mapped to Homo sapiens genome by STAR. Uniquely mapped reads which with "NH: i:1" flag were retained for further analysis by SAMtools and PCR duplicate reads were removed by in-house script based on sharing identical random sequence. To quantify gene expression and translation abundance, we counted sequenced reads mapping to coding sequence (CDS) region by featureCounts.
genome build: Ensembl GRCh38.p5
processed data files format and content: raw counts

extract_protocol:
500 µl lysate was prepared as described. 100 µl lysate was used to prepare total RNA library, meanwhile, ribosome footprinting and subsequent library preparation of ribosome protected RNA fragments (RPFs) was performed with 400 µl lysate. Add 10 U RNase I to 400 µl lysate and incubate for 45 min at room temperature with gentle mixing. Then the nuclease digestion was stopped by adding 1 µl RNase inhibitor. RPFs were purified with MicroSpinS-400 columns (GE Healthcare Life Sciences) followed by size selection, which was conducted using 15% TBE-urea gel. rRNAs were depleted with the NEBNext rRNA depletion kit (New England BioLabs). Following end repair and 3'adaptor ligation, RNAs were reverse transcribed using SuperScript III (Thermo Fisher, USA).
cDNA libraries were prepared as described (Nat Protoc. 2012 Jul 26;7(8):1534-50) and sequenced with Illumina HiSeq X Ten platform.

treatment_protocol: For Ribo-seq and RNA-seq: K562 cells were incubated with 100 µg /ml cycloheximide (CHX, Sigma) at 37°C for 10 min, washed twice with ice-cold PBS containing 100 µg/ml of CHX and lysed in lysis buffer.

value: molnc301b_m2 overexpression

value: Ribo-seq


title: RPF_molnc301b_del_rep1

description: Ribo-seq after molnc301b_del overexpression

value: molnc301b_del overexpression


title: Case 4: Central giant cell granuloma of the jaws


title: Case 6: Central giant cell granuloma of the jaws


title: Case 5: Central giant cell granuloma of the jaws


title: Case 3: Central giant cell granuloma of the jaws


title: RPF_molnc301b_m2_rep1


title: Case 16: Cherubism

description: Cherubism


title: M3-Right-03-Neuron-RNA-R1

description: MOWChIP-seq data. Name format: Mouse number-Left/right hemisphere-Slice number-Neuron/glia-Marker-Replicate number


title: K562_library1_2

description:
scRNA-seq with molncRNA(-) and internal control gRNAs, 10X lane 2
Sample 2


title: M3-Right-02-Neuron-RNA-R2


title: Case 2: Central giant cell granuloma of the jaws


title: Case 1: Central giant cell granuloma of the jaws


title: Case 12: Cherubism

description: Cherubism


title: RPF_molnc301b_m1_rep1

description: Ribo-seq after molnc301b_m1 overexpression

value: molnc301b_m1 overexpression


title: RPF_molnc301b_wt_rep2

description: Ribo-seq after molnc301b_wt overexpression

value: molnc301b_wt overexpression


title: M3-Right-02-Neuron-RNA-R1


title: RPF_molnc301b_del_rep2


title: Case 9: Central giant cell granuloma of the jaws


title: M3-Right-05-Neuron-RNA-R1


title: H3-NHS-r3

city: Potsdam

first: Isabel

last: Bäurle

address: Karl-Liebknecht-Str. 24-25

department: Institut für Biochemie und Biologie

institute: Universität Potsdam

data_processing:
Reads were mapped against the Arabidopsis thaliana reference genome (TAIR10) using bwa mem.
Duplicates were removed using samtools markdup
Reads for biological replicates were merged. Both individual samples and merged replicates were processed as follows.
Normalized coverage tracks were generated using deeptools bamCoverage (--binSize 10 --normalizeUising RPGC --extendReads). Artifactual regions identified using Greenscreen, chloroplast and mitochondria sequences were excluded.
Assembly: Arabidopsis thaliana TAIR10
Supplementary files format and content: Normalized coverage files in bigWig format

organism: Arabidopsis thaliana

source_name: seedlings

growth_protocol: Plants were grown on GM medium (1% glucose) under a 16/8 h light/dark cycle at 23/21°C.

extract_protocol:
Seedlings were crosslinked under vacuum in PBS buffer with 1 % (w/v) formaldehyde for 10 min on ice. Chromatin was extracted as described previously (Kaufmann et al., 2010). Chromatin was fragmented using a Bioruptor (Diagenode – 20 cycles 30sec on/30sec off). For single ChIP, chromatin was immunoprecipitated overnight at 4°C with IgG (ThermoFisher 026102), anti-panH3 (Diagenode C15200011) or anti-GFP (Chromotek PABG1) antibodies. Antibody-chromatin complexes were purified with Dynabeads (ThermoFischer 10002D) and successively washed with low salt (150 mM NaCl, 1 % (v/v) Triton X-100), high salt (500 mM NaCl, 1 % (v/v) Triton X-100), LiCl buffer (250 mM LiCl, 1 % (v/v) NP-40) and TE buffer. Chromatin was eluted with single ChIP elution buffer (1 % (w/v) SDS, 0.1 M NaHCO3).
ChIP DNA was generated and libraries were prepared using NEBNext Ultra II FS DNA Library preparation kit (NEB E7645). Quality control was performed with TapeStation D1000 ScreenTape system (Agilent) and a Qubit fluorometer (Thermo Fisher).

treatment_protocol: HS treatments were performed on 5 d-old seedlings by a treatment of 37°C for 60 min, 22°C for 90 min and 44°C for 45 min.

value: seedlings

tag: chip antibody

value: No Heat Shock


title: M3-Right-04-Neuron-RNA-R1


title: H3-NHS-r1


title: M3-Right-03-Neuron-RNA-R2


title: M3-Right-01-Neuron-RNA-R2


title: RPF_molnc301b_wt_rep1


title: RPF_molnc301b_m1_rep2


title: RPF_Ctrl_rep2

description: control Ribo-seq

value: empty vector


title: H3-4h-r3


title: H3-4h-r1


title: M3-Right-01-Neuron-RNA-R1


title: GFP-NHS-r2

value: anti-GFP


title: H3-4h-r2


title: RPF_Ctrl_rep1

description: control Ribo-seq


title: RNA_molnc301b_del_rep2

description: RNA-seq after molnc301b_del overexpression

data_processing:
For Ribo-seq and RNA-seq: Raw reads were trimmed by Cutadapt. Then, reads with distinct inline barcodes were demultiplexed using in-house scripts, and the 10-mer random sequence was appended to the reads name in bam file for later usage. Low quality reads were filtered by trimmomatic with parameter "SLIDINGWINDOW:4:15". In Ribo-seq library, reads length in 25-50 bp were retained for subsequent analysis. rRNA reads were removed by aligning reads with rRNA reference sequence on NCBI database by bowtie. Cleaned reads were mapped to Homo sapiens genome by STAR. Uniquely mapped reads which with "NH: i:1" flag were retained for further analysis by SAMtools and PCR duplicate reads were removed by in-house script based on sharing identical random sequence. To quantify gene expression and translation abundance, we counted sequenced reads mapping to coding sequence (CDS) region by featureCounts.
genome build: Ensembl GRCh38.p5
processed data files format and content: raw counts

value: RNA-seq


title: GFP-NHS-r1


title: GFP-4h-r2


title: GFP-4h-r3


title: H3-NHS-r2


title: M2-Right-20-Neuron-H3K27me3-R1

value: brain


title: GFP-4h-r1


title: M2-Right-20-Glia-H3K27me3-R2


title: H3-NTC-3

value: NHS


title: M2-Right-20-Glia-H3K27me3-R1


title: H3-NTC-2


title: RNA_molnc301b_m2_rep2

description: RNA-seq after molnc301b_m2 overexpression


title: RNA_molnc301b_del_rep1


title: M2-Right-19-Neuron-H3K27me3-R2


title: GFP-NHS-r3


title: M2-Right-20-Neuron-H3K27me3-R2


title: RNA_molnc301b_m2_rep1


title: RNA_molnc301b_m1_rep2

description: RNA-seq after molnc301b_m1 overexpression


title: H3-1d-2


title: H3-1d-3


title: M2-Right-19-Neuron-H3K27me3-R1


title: H3-1d-1


title: GFP-NTC-3


title: GFP-NTC-2


title: RNA_molnc301b_m1_rep1


title: M2-Right-19-Glia-H3K27me3-R2


title: GFP-1d-3


title: GFP-NTC-1


title: GFP-1d-2


title: M2-Right-19-Glia-H3K27me3-R1


title: M2-Right-18-Neuron-H3K27me3-R2


title: RNA_molnc301b_wt_rep1

description: RNA-seq after molnc301b_wt overexpression


title: Output fast-growing Replicate 2

city: Basel

first: Alexandra

middle: Michaela

last: Bendel

address: Maulbeerstr. 66

department: Genome Regulation

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

data_processing:
For the DMS, barcode reads were matched to the expected barcode sequences, aggregated by amino acid substitutions and processed with mutscan
Supplementary files format and content: csv file. Contains counts
Supplementary files format and content: csv file. Contains average growth rates

organism: Saccharomyces cerevisiae

extract_protocol:
DNA was extracted using a custom yeast Mini- or Midiprep protocol
Sequencing libraries were constructed by PCR with primers cotaining the illumina adapters


title: GFP-1d-1


title: RNA_Ctrl_rep2

description: control RNA-seq


title: M2-Right-18-Neuron-H3K27me3-R1


title: Output slow-growing Replicate 2


title: M2-Right-18-Glia-H3K27me3-R2


title: Output balanced Replicate 2


title: OE_molnc301b_del_rep2

description: Poly(A)+ RNA-seq after molnc301b_del overexpression on day11 of erythroid differentiation

data_processing:
For Poly(A)+ RNA-seq on day11 of erythroid differentiation: Analysis of RNA-seq data, reads were aligned to Homo sapiens genome using Tophat2 PE mode with the default parameter, and uniquely mapped reads were retained for further analysis filtered by SAMtools. HTSeq-counts or featureCounts were used for calculating annotated gene counts. Differential gene expression analysis was conducted using DESeq2 with the P-value<0.05, and all genes with non-zero counts in the samples were considered.
genome build: Ensembl GRCh38.p5
processed data files format and content: raw counts

source_name: CD34+ cell

growth_protocol: For Poly(A)+ RNA-seq on day11 of erythroid differentiation: CD34+ cells cultured in IMDM medium supplemented with 20% BIT,5ng/ml IL-3, 100 ng/ml SCF ,10^-6 M Hydrocortisone and 3 U/ml human EPO for 8 days. From days 8 through 14, SCF and EPO were included in the culture medium, and only EPO was included during days 14 to 18.

extract_protocol:
The cells were collected and RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: The harvested viral particles were added into the HSPCs plus with 8 µg/ml polybrene for 12 hours. Then the cells were replaced with fresh complete medium and subjected to the following experiments.

value: cord blood CD34+ derived erythroid cells

value: Poly(A)+ RNA-seq


title: Output fast-growing Replicate 1


title: RNA_Ctrl_rep1

description: control RNA-seq


title: Output balanced Replicate 1


title: M2-Right-18-Glia-H3K27me3-R1


title: OE_molnc301b_del_rep1


title: Output slow-growing Replicate 1


title: M2-Right-17-Neuron-H3K27me3-R2


title: H3-NTC-1


title: M2-Right-17-Neuron-H3K27me3-R1


title: RNA_molnc301b_wt_rep2


title: Input balanced Replicate 2


title: Input fast-growing Replicate 2


title: Input slow-growing Replicate 2


title: OE_molnc301b_m2_rep2

description: Poly(A)+ RNA-seq after molnc301b_m2 overexpression on day11 of erythroid differentiation


title: Input balanced Replicate 1


title: M2-Right-17-Glia-H3K27me3-R2


title: M2-Right-17-Glia-H3K27me3-R1


title: OE_molnc301b_m1_rep2

description: Poly(A)+ RNA-seq after molnc301b_m1 overexpression on day11 of erythroid differentiation


title: OE_molnc301b_m1_rep1


title: Set1A_input

city: wuhan

first: Yu

last: Qi

state: Hubei

address: xujiapeng

department: College of life sciences

institute: Hubei university

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to sacCer3 whole genome using bowtie v0.12.2 with parameters -q -p 5 -e 100 -y -a -m 10 --best --strata
Assembly: sacCer3
Supplementary files format and content: bigwig

source_name: genomic DNA

growth_protocol: Cells were grown in YPD until mid-log phase

extract_protocol:
Lysates were clarified from sonicated yeast cell pellets and histone-DNA complexes were isolated with indicated antibodies.
Libraries were prepared using the HTP KAPA kit

treatment_protocol: No treatment

value: genomic DNA

value: none (INPUT)


title: Set1F_input


title: Set1F_IP

value: FLAG (F1804-1MG; Sigma-Aldrich)


title: Set1A_IP


title: M2-Right-16-Neuron-H3K27me3-R2


title: S288E_H3K4me3

data_processing:
Fastq files for chip-seq samples were generated with bcl2fastq 1.8.3 from Illumina.
ChIP-seq reads were trimmed from 5' end eith at least 6 bp until the final base had a quality score > 30, using Trim Galore , discarding reads left with < 70 bp
ChIP-seqdatawasalignedtosacCer3genomefromUCSCwithbowtie2(2.2.0)using-k1
bigWig files were created by using deeptool bamCompare command, normalizing to reads per kilomillion by multiplying by 1 kilomillion and dividing by the total number of alignments for each sample.
Peask were called by using Macs2 callpeak command with -g 1.2e-7 --nomodel AND with the significance cut-off q-value <=0.01
Assembly: sacCer3
Supplementary files format and content: bigWig

growth_protocol: Cells were grown in YPD  medium until mid-log phase

extract_protocol:
Lysates were clarified from sonicated yeast cell pellets and histone-DNA complexes were isolated with antibody against  FLAG (F1804-1MG; Sigma-Aldrich),
ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.

value: H3K4me3 (Abclonal; A2357)


title: S288E_H3

value: H3 (Abcam; ab1791)


title: M2-Right-16-Neuron-H3K27me3-R1


title: OE_molnc301b_wt_rep1

description: Poly(A)+ RNA-seq after molnc301b_wt overexpression on day11 of erythroid differentiation


title: S288A_H3


title: S288A_H3K4me3


title: WT_H3 (Set1 control)


title: M2-Right-16-Glia-H3K27me3-R2


title: Input fast-growing Replicate 1


title: M2-Right-16-Glia-H3K27me3-R1


title: WT_H3K4me3


title: Ctrl_rep4

description: control Poly(A)+ RNA-seq on day11 of erythroid differentiation


title: M2-Right-15-Neuron-H3K27me3-R2


title: Ctrl_rep3


title: Input slow-growing Replicate 1


title: Cla4_input


title: OE_molnc301b_m2_rep1

value: molnc301b_m2 overexpression


title: Ctrl_rep2


title: Cla4_IP


title: M2-Right-15-Neuron-H3K27me3-R1


title: WT TP 2

city: Munich

first: Vanessa

last: Arfelli

state: Bavaria

address: Max-Lebsche-Platz 30

department: Medical Clinic and Polyclinic III

institute: Munich University Hospital, Ludwig-Maximilians University (LMU)

description: Libraries were prepared using Chromium Next GEM Single Cell 3' GEM Kit v3.1 (10x Genomics).

data_processing:
Sample demultiplexing and alignment of the data were done using the Cell Ranger software (v6.1.2) and the human genome GRCh38.p13 from Gencode (release 39).
Further data processing (quality control, filtering, normalisation, highly variable gene selection, embedding and visualisation) was performed using the python package scanpy v1.9.1. (Wolf et al, 2018).
We filtered cell barcodes with less than 3000 counts, more than 100,000 counts or more than 20% of mitochondrial reads. Additionally, we removed cells with less than 600 genes captured.
Cell type annotation was done using the package celltypist v1.3.1. (Domínguez Conde et al, 2022).
Supplementary files format and content: HDF5 Feature-Barcode Matrix Format. Binary format of multidimensional arrays containing count of reads containing valid barcodes and UMIs as well as metadata on the libraries and gene sets/annotations.

source_name: bone marrow, hematopoietic progenitor cells

growth_protocol: CD34+ bone marrow cells (Lonza, Basel, Switzerland) were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Gibco, Carlsbad, CA, USA), supplemented with 20% FBS, 1% penicillin-streptomycin, 4 µM L-Glutamine (Gibco), 10 ng/ml interleukin (IL)-3, 20 ng/ml IL-6, 20 ng/ml Flt3-L, 20 ng/ml GM-CSF, 20 ng/ml stem cell factor (SCF), 20 ng/ml thrombopoietin (TPO) (cytokines were obtained from Peprotech, Hamburg, Germany).

extract_protocol:
At day 30 and day 60 of the competitive growth culture, cells were harvested and a cell suspension of 16000 cells (multiplet rate 7,6%, recovery of 10000 cells) was used for library preparation.
Chromium Next GEM Single Cell 3' GEM Kit v3.1 (10x Genomics, Pleasanton, CA, USA)

treatment_protocol: Human CD34+ bone marrow cells were double transduced with retroviral particles carrying CSF3R WT or CSF3R T618I (GFP) together with RUNX1–RUNX1T1tr (tdTomato). Expansion of single and double fluorescent marker-positive cells was then followed by flow cytometry over 60 days after transduction as described before (Wichmann et al., 2015).

tag: cell marker

value: RUNX1-RUNX1T1 + CSF3R WT

tag: transduction

value: Double transduction with retroviral particles carrying the RUNX1-RUNX1T1 truncated form and CSF3R WT.

tag: time

value: Day 60


title: T618I TP 2

value: bone marrow, hematopoietic progenitor cells

value: RUNX1-RUNX1T1 + CSF3R T618I

value: Double transduction with retroviral particles carrying the RUNX1-RUNX1T1 truncated form and CSF3R T618I.


title: OE_molnc301b_wt_rep2

value: molnc301b_wt overexpression


title: T618I TP 1

value: Day 30


title: M2-Right-15-Glia-H3K27me3-R2

value: brain


title: WT TP 1


title: shCtrl_rep2

description: sh-control Poly(A)+ RNA-seq

data_processing:
For Poly(A)+ shRNA-seq in K562 cell line: Analysis of RNA-seq data, reads were aligned to Homo sapiens genome using Tophat2 PE mode with the default parameter, and uniquely mapped reads were retained for further analysis filtered by SAMtools. HTSeq-counts or featureCounts were used for calculating annotated gene counts. Differential gene expression analysis was conducted using DESeq2 with the P-value<0.05, and all genes with non-zero counts in the samples were considered.
genome build: Ensembl GRCh38.p5
processed data files format and content: raw counts

extract_protocol:
For Poly(A)+ shRNA-seq in K562 cell line: The cells were collected and RNA was harvested using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: sh-control vector

value: K562 cell line


title: Ctrl_rep1

value: empty vector

value: cord blood CD34+ derived erythroid cells

value: Poly(A)+ RNA-seq


title: T618I 2-6 TP 2

description: Libraries were constructed using a bulk RNA-seq method named Prime-seq, which uses direct cell lysis, bead cleanup, cDNA synthesis and pre-aplification with KAPA HiFi Polymerase (see Janjic et al, 2022)

data_processing:
Raw Fastq data were processed with the pipeline zUMIs, version 0.2.56b (Parekh et al., 2018)
cDNA reads were mapped to the human genome hg38 using the software STAR version 2.6.0a (Dobin et al., 2013).
Gene annotations were obtained from Ensembl GRCh38.84 gft.
Differential expression was assessed using edgeR/limma package (Ritchie et al., 2015; Robinson et al., 2010).
Supplementary files format and content: Sample (columns) and gene (rows) -wise UMI expression table

extract_protocol:
After 60 days of the competitive growth culture, 1x104 cells were harvested and washed with DPBS 1x once, and then resuspended in 100 µl of Buffer RLT Plus, RNeasy Plus Lysis Buffer (Qiagen, Hilden, Germany) supplemented with 1% b-mercapoethanol.
Prime-seq (Janjic et al, 2022 - https://doi.org/10.1186/s13059-022-02660-8)

treatment_protocol: Human CD34+ bone marrow cells were double transduced with retroviral particles harboring CSF3R WT, CSF3R T618I or empty vector  (MIG, marked with GFP) together with RUNX1–RUNX1T1tr (marked with tomato). Expansion of single and double fluorescent marker-positive cells was then followed by flow cytometry over 60 days after transduction as described before (Wichmann et al., 2015).

value: Double transduction with retroviral particles carrying the coding sequences of RUNX1-RUNX1T1 truncated form and CSF3R T618I.


title: T618I 2-4 TP 2


title: T618I 2-5 TP 2


title: shCtrl_rep1

description: sh-control Poly(A)+ RNA-seq


title: M2-Right-14-Neuron-H3K27me3-R2


title: M2-Right-15-Glia-H3K27me3-R1


title: T618I 2-2 TP 2


title: HSPCs_day18_RNA_rep2

data_processing:
For Poly(A)+ RNA-seq during erythroid differentiation: Analysis of RNA-seq data, reads were aligned to Homo sapiens genome using Tophat2 PE mode with the default parameter, and uniquely mapped reads were retained for further analysis filtered by SAMtools. HTSeq-counts or featureCounts were used for calculating annotated gene counts. Differential gene expression analysis was conducted using DESeq2 with the P-value<0.05, and all genes with non-zero counts in the samples were considered.
genome build: Ensembl GRCh38.p12
processed data files format and content: raw counts

growth_protocol: For Poly(A)+ RNA-seq during erythroid differentiation: CD34+ cells cultured in IMDM medium supplemented with 20% BIT,5ng/ml IL-3, 100 ng/ml SCF ,10^-6 M Hydrocortisone and 3 U/ml human EPO for 8 days. From days 8 through 14, SCF and EPO were included in the culture medium, and only EPO was included during days 14 to 18.

extract_protocol:
CD34+ drived erythroid cells cells were collected and RNA was harvested at indicated days using Trizol reagent.
RNA libraries were prepared for sequencing using standard Illumina protocols.


title: T618I 1-6 TP 2


title: T618I 2-1 TP 2


title: T618I 1-5 TP 2


title: M2-Right-14-Neuron-H3K27me3-R1


title: M2-Right-14-Glia-H3K27me3-R2


title: HSPCs_day18_RNA_rep1

description: Poly(A)+ RNA-seq during erythroid differentiation


title: T618I 1-4 TP 2


title: HSPCs_day14_RNA_rep2


title: M2-Right-14-Glia-H3K27me3-R1


title: M2-Right-13-Neuron-H3K27me3-R2


title: T618I 1-3 TP 2


title: T618I 1-2 TP 2


title: HSPCs_day11_RNA_rep2


title: WT 2-5 TP 2

value: Double transduction with retroviral particles carrying the coding sequences of RUNX1-RUNX1T1 truncated form and CSF3R WT.


title: T618I 1-1 TP 2


title: HSPCs_day14_RNA_rep1


title: WT 2-4 TP 2


title: HSPCs_day11_RNA_rep1


title: WT 2-6 TP 2


title: M2-Right-13-Neuron-H3K27me3-R1


title: WT 2-2 TP 2


title: WT 1-6 TP 2


title: WT 2-1 TP 2


title: T618I 2-3 TP 2


title: molnc301b_IP_odd

description: ChIRP-seq of molnc-301b in K562 cell line

data_processing:
For ChIRP-seq: Reads were aligned to Homo sapiens genome using Bowtie2. Only reads with mapping quality score >30 were retained for further analysis by SAMtools. And per-base coverage was normalized in accordance with previous studies. For each base pair of the genome, true coverage of this base in this sample was defined as the minimum coverage of the even lane and odd lane and a SAM file was generated for peak calling. Peaks of each sample were then called using MACS2 against its corresponding input. We filtered for peaks with substantial correlation of the raw data profile, and high coverage across the peaks were accepted. For our ChIRP-seq sample, thresholds of average coverage >0.25, fold enrichment >5 and fold differences >1. These peaks were annotated by Homer.
genome build: Ensembl GRCh38.p5
processed data files format and content: peak text file

growth_protocol: For ChIRP-seq: Human erythroleukemia cell line K562 was maintained in RPMI1640 supplemented with 10% FBS (Gibco, Carlsbad, CA, USA).

value: ChIRP-seq


title: molnc301b_Input


title: M2-Right-13-Glia-H3K27me3-R2

description: MOWChIP-seq data. Name format: Mouse number-Left/right hemisphere-Slice number-Neuron/glia-Marker-Replicate number

data_processing:
Raw reads were trimmed with TrimGalore 0.4.1
Trimmed reads were aligned using bowtie 1.1.2. Uniquely mapped reads were filtered for known blacklisted genome regions using Samtools and bedtools to remove ChIP-seq artifacts.
Reads were extended by 100 bp on either ends
Sample was normalized and input subtracted. The reference genome was then divided into 100-bp bins and ChIP-seq signal for each bin was counted for both ChIP and input samples.
Sample was converted to BigWig format
Assembly: mm10
Supplementary files format and content: Normalized ChIP-seq and RNA-seq signal in BigWig format


title: molnc301b_IP_even


title: WT 1-3 TP 2

value: RUNX1-RUNX1T1 + CSF3R WT


title: WT 1-4 TP 2


title: M2-Right-12-Neuron-H3K27me3-R1


title: WT 1-2 TP 2


title: M2-Right-12-Neuron-H3K27me3-R2


title: M2-Right-13-Glia-H3K27me3-R1


title: WT 1-1 TP 2


title: EV 2-6 TP 2

value: RUNX1-RUNX1T1 + empty vector control

value: Double transduction with retroviral particles carrying the RUNX1-RUNX1T1 truncated form and empty vector (MIG).


title: WT 2-3 TP 2

value: bone marrow, hematopoietic progenitor cells

value: Day 60


title: M2-Right-12-Glia-H3K27me3-R1


title: EV 2-5 TP 2


title: DM3

city: Fuzhou

first: jianzhang

last: Hu

address: Fuzhou

institute: Fujian Medical University

data_processing:
After quality control, the reads were 3’5'-adaptor trimmed and filtered 15 bp reads with cutadapt software.
The trimmed reads were aligned to reference genome with bowtie software.
For each sample, known miRNAs, and predicted novel miRNAs were identified and enumerated using miRDeep2 software package (Freedlander et al. Nature Biotechnology, 26, 407-415 (2008) ).
Raw reads were normalized by calculated CPM value.
Assembly: miRBase Release 22
Supplementary files format and content: The xlsx format file contains the CPM value of gene in every sample.

source_name: The trigeminal ganglion of diabetic mice

extract_protocol:
Total RNA was extracted using the TRIzol reagent (Invitrogen/Life Technologies) following the manufacturer’s protocol. RNA concentration was quantitated using the NanoDrop ND-100 Spectrophotometer (NanoDrop Technologies, Wilmington, DE).Small RNA libraries were constructed using New England Biolabs (NEB) NEBNext Multiplex Small RNA Library Prep Set for Illumina sequencers and the NEB standard protocol. Individual libraries were constructed using 1µg of total RNA isolated from each sample.
1) 3’-adaptor ligation;2) 5’-adaptor ligation;3) cDNA synthesis;4) PCR amplification;5) size selection of 135   155 bp PCR amplified fragments (corresponding to 15   35 nt small RNAs).

treatment_protocol: Diabetic mice were injected intraperitoneally with low-dose streptozotocin (STZ, 50 mg/kg; Sigma‒Aldrich, USA) for 5 days, and then random intravenous blood glucose concentrations were measured every 4 weeks.

value: trigeminal ganglion

tag: group

value: diabetic


title: EV 2-4 TP 2


title: M2-Right-11-Neuron-H3K27me3-R2


title: DM1


title: EV 2-3 TP 2


title: WT 1-5 TP 2


title: EV 1-6 TP 2


title: EV 2-2 TP 2


title: EV 2-1 TP 2


title: M2-Right-11-Neuron-H3K27me3-R1


title: Ctrl4

source_name: The trigeminal ganglion of normal mice

value: normal


title: EV 1-5 TP 2


title: EV 1-3 TP 2


title: Ctrl2


title: Ctrl3


title: M2-Right-11-Glia-H3K27me3-R2

value: brain


title: M2-Right-12-Glia-H3K27me3-R2


title: EV 1-1 TP 2


title: M2-Right-11-Glia-H3K27me3-R1


title: EV 1-4 TP 2


title: B2M exon1 - RNP - Rep2

city: Milan

first: Stefano

last: Beretta

address: Via Olgettina, 60

country: Italy

institute: San Raffaele Telethon Institute for Gene Therapy

data_processing:
Data analyzed withthe CRISPResso2 pipeline:
reads were trimmed using the Trimmomatic
reads were aligned against the reference vector
quantification around the guide cut site was performed for each found allele
Assembly: Custom reference vector sequences and sgRNAs
Supplementary files format and content: Tab-separated results of the different allele found with corresponding quantifications

source_name: Cutured HSPC

extract_protocol:
gDNA was isolated with QIAamp DNA Micro Kit (QIAGEN) according to the manufacturer's instructions.
Custom library construction protocol. PCR amplicons for each individual sample were generated by nested PCR starting from >50-100 ng of purified gDNA.The first PCR step was performed using the following amplification protocol: 95°C x 10’, (95°C x 30’’, 59°C x 30’’, 72°C x 20’’) x 20 cycles, 72°C x 10’. The second PCR step was performed using the same reagents of the first step, 5 μl of the PCR product and the following amplification protocol: 95°C x 10’, (95°C x 30’’, 59°C x 30’’, 72°C x 20’’) x 20 cycles, 72°C x 10’. Primers used for the second PCR step contained P5/P7 sequences, i5/i7 Illumina tags to allow multiplexed sequencing and R1/R2 primer binding sites. The PCR amplicon from each sample was separately purified by QIAquick PCR Purification Kit (QIAGEN). Concentration and quality of amplicons were assessed by QuantiFluor ONE dsDNA system and 4200 Tapestation System (Agilent). Amplicons from up to 25 differently tagged samples were multiplexed at equimolar ratios.
Other: amplicon sequencing

value: Cutured HSPC

tag: target locus

value: B2M exon1

value: RNP


title: DKO GSK343 Rep3

city: London

first: Frederick

middle: Andrew

last: Dick

state: ON

address: 790 Commissioners Road E

department: Pathology

country: Canada

institute: Western University

data_processing:
Base calls and demultiplexing were performed using bcl2fastq
Gene expression analysis: Reads were mapped to mm10 with STAR/2.7.8a and GENCODE VM22 comprehensive gene annotation. SAM files were converted to BAM, sorted and indexed with samtools/1.12. Reads aligned to gene annotations were counted with htseq-count/0.11.0 using default settings. Count tables were imported into Rstudio using R/3.6.3. Differential gene expression analysis was performed with edgeR/3.28.1.
Repetitive element expression analysis: Reads were mapped to mm10 with bowtie2/2.3.3.1 using default settings. Unique and multimapped reads were separated using RepEnrich2. RepEnrich2 was used to count the number of reads aligning to each repName element in a custom repeat annotation file. The annotation was generated by removing low complexity and simple repeat repClass elements from Repeat Masker table downloaded from UCSC Table Browser. Any element overlapping mm10 blacklist coordinates (ENCODE) was also removed. Count tables were imported into Rstudio using R/3.6.3. Differential gene expression analysis was performed with edgeR/3.28.1.
Assembly: mm10
Supplementary files format and content: csv: count table of gene expression; txt: count table of repetitive element expression

source_name: B16-F10 melanoma cells

growth_protocol: B16-F10 cells (3e5) were treated with either DMSO or  5 uM GSK343 for 4 days in DMEM (Wisent) supplemented with 10% (v/v) FBS, 100 IU penicillin, 100 ug/mL streptomycin, 2 mM glutamine. Culture medium was changed daily with fresh vehicle or GSK343.

extract_protocol:
Total RNA was extracted using Monarch Total RNA Miniprep kit (NEB T2010S)
Libraries were prepared with VAHTS Total RNA-seq library prep kit (GeneBio Systems, NR603-01), following the manufacturer's recommendations

treatment_protocol: DMSO or 5 uM GSK343 for 4 days

value: Rigi/Cgas double KO

value: 5 uM GSK343

tag: molecule type

value: rRNA-depleted RNA


title: DKO GSK343 Rep2


title: DM4

tag: tissue


title: M2-Right-10-Neuron-H3K27me3-R2

extract_protocol:
Nuclei extraction from dissected brain tissue was performed using previously described protocol with minor modifications. All steps were conducted on ice and centrifugation was conducted at 4 ℃. A neocortex slice was thawed on ice and transferred to a glass tissue homogenizer (Sigma, d9036-1SET). 3 ml of ice-cold nuclei extraction buffer was added [0.32 M sucrose, 5mM CaCl2, 2 mM Mg(Ac)2 , 0.1 mM EDTA, 10 mM tris-HCl, 0.1% (v/v) Triton X-100, freshly added 1% protease inhibitor cocktail (PIC, Sigma-Aldrich), 0.1 mM PMSF, 1 mM DTT, and 0.06 U/µl ribonuclease (RNase) inhibitor (2313A,Takara Bio, needed when mRNA extraction was performed)]. The tissue was homogenized with 10 times of slow douncing using a loose pestle and 15 times of douncing using a tight pestle (D9063, Sigma-Aldrich). The homogenate was filtered through a 40-µm cell strainer (22363547, Thermo Fisher Scientific) into a 15-ml centrifuge tube and centrifuged at 1000 rcf for 10 min at 4 ℃. Supernatant was discarded and the pellet was resuspended in 0.5 ml of ice-cold nuclei extraction buffer. 0.75 ml of 50% (w/v) iodixanol (made by mixing 4 ml of OptiPrepTM gradient (Sigma-Aldrich) and 0.8 ml of diluent [150 mM KCl, 30 mM MgCl2, and 120 mM tris-HCl]) was added. The suspension was gently mixed by inverting the tube up and down 5 times and then centrifuged at 10,000 rcf for 20 min. Supernatant was carefully removed and the pellet incubated with 100 µl of 2% (w/v) normal goat serum (50062Z, Life Technologies) in Dulbecco’s PBS (DPBS, Life technologies) on ice. The pellet was then resuspended and transferred into a new Eppendorf tube with an addition of 200 µl of 2% normal goat serum. To differentiate neurons from glia, 10 µl of 2 ng/µl Anti-NeuN antibody conjugated with Alexa 488 (MAB377X, EMD Millipore) was mixed with the suspension. The mixture was incubated at 4 ℃ for 1 h on a rotator. The nuclei solution was sorted using FACS (BS FACSAria, BD Bioscience) to separate neuronal (NeuN+) and glial (NeuN-) fractions.   Chromatin fragmentation:  M1-2: 4,000 nuclei per replicate was used so that both neuronal and glial fractions of every neocortex slice sample yielded enough material for two ChIP-seq replicates. The nuclei suspension (containing 8,000 nuclei) was topped up to 1 ml using Dulbecco’s PBS (DPBS; 14190144, Life Technologies). 200 µl of 1.8M sucrose, 10 µl of 1M CaCl2, and 3 µl of 1M Mg(Ac)2 were then added sequentially. The sample was mixed gently by inverting the tube up and down and then incubated for 15 min on ice. The suspension was centrifuged at 3,000 rpm for 15 min at 4 ℃. Supernatant was carefully removed, and the pellet was resuspended in 10 µl DPBS. 0.1 µl of PIC, 0.1 µl of 100 mM PMSF and 10 µl of lysis buffer [4% Triton X-100, 100 mM tris (pH 7.5), 100 mM NaCl, and 30 mM MgCl2] were added, mixed by vortexing and then incubated at room temperature for 10 min. 1 µl of 0.1 M CaCl2 and 0.4 µl of 100 U/µl MNase (88216, Thermo Fisher Scientific) were then added, mixed by vortexing and incubated at room temperature for 10 min. 2.22 µl of 0.5 M EDTA (pH 8) was added to quench the rection. The sample was vortexed and incubated on ice for 10 min before it was centrifuged at 16,100 g for 5min. The supernatant (~20 µl, containing chromatin) was transferred to a new tube and 60 µl of IP buffer [20 mM Tris-HCl, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100] was added to obtain a total volume of ~80 µl. The solution was finally split for two ChIP-seq replicates. The chromatin was stored on ice before loaded into the MOWChIP device.  M4-5: 200 µl of sorted neuronal nuclei suspension from one neocortex slice sample was used to prepare chromatin (containing ~30,000 nuclei). 2 µl of PIC, 2 µl of 100 mM PMSF and 200 µl of lysis buffer were added, mixed by vortexing and incubated at room temperature for 10 min. 4 µl of 1 M CaCl2 and 5 µl of 100 U MNase (88216, Thermo Fisher Scientific) were added, mixed by vortexing and incubated at room temperature for 10 min. 44 µl of 0.5 M EDTA was then added, mixed by vortexing and incubated on ice for 10 min. The mixture was finally centrifuged at 16,100g for 5 min at 4 °C. Supernatant containing fragmented chromatin (~ 440 µl) was collected into a new 1.5 ml tube and placed on ice before use. 70 µl of chromatin (containing chromatin from ~4,000 nuclei) was used for each MOWChIP-seq assay for consistency with data on M1-2.  Antibody coating on IP beads:  5 µl of Dynabeads Protein A (10002D, Thermo Fisher Scientific) were used in each MOWChIP assay. Beads were washed twice with 150 µl of IP buffer [20 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% (w/v) sodium deoxycholate, 0.1% SDS, 1% (v/v) Triton X-100] and then resuspended in 150 µl of IP buffer. An antibody that targets a specific modified histone was added to coat the beads: anti-H3K27ac (ab4729, Abcam) at 0.125 µg/assay or anti-H3K27me3 (39155, Active Motif) at 0.5 µg/assay. The bead/antibody suspension was incubated on a rotator at 4 °C overnight. Beads were then washed with IP buffer twice and resuspended in 5 µl of IP buffer for use in MOWChIP.  MOWChIP:  The MOWChIP process, including device fabrication and operation, was performed following our published protocol (Zhu, B., Hsieh, YP., Murphy, T.W. et al. MOWChIP-seq for low-input and multiplexed profiling of genome-wide histone modifications. Nat Protoc 14, 3366–3394 (2019).).
ChIP-seq libraries:  ~4,000 neuronal or glial nuclei were used to produce each ChIP-seq library and two technical replicates were generated on each neocortex slice sample. The input libraries were created using the same starting quantity of nuclei (~ 4,000 per library) with the MOWChIP step skipped. Libraries were constructed using 2S Plus DNA Library kit (10009877, IDT) and 2S CDI Adapter S1 indexing kit (10009895, IDT) following the manufacturer’s instructions. 5% (v/v) EvaGreen (31000, Biotium) was added to the amplification mixture to monitor the progress of library amplification. Amplification was stopped once an increase of 2,000 RFU was reached. Library was eluted to 10 µl of low EDTA TE buffer.  mRNA-seq:  mRNA libraries were prepared following Smart-seq2 protocol55 with minor modifications. ~7,500 neuronal nuclei (from M3) were used to generate each RNA-seq library, and each slice sample has two technical replicates. 50 μl of neuronal suspension containing about 7,500 nuclei was used for RNA extraction using RNeasy Mini kit (QIAGEN) and RNase-Free DNase Set (79254, QIAGEN). Extracted RNA was concentrated using ethanol precipitation and resuspended in 5 μl of RNase-free water. 4.6 μl of RNA solution, 2 μl of 10 μM oligo-dT primer and 2 μl of 10 mM dNTP mix were mixed, incubated at 72°C for 3 mins and then stored on ice. 11.4 μl of reverse-transcription mix (made from 1 μl of SuperScript II reverse transcriptase (200 U/μl), 0.5 μL of RNase inhibitor (40 U/μl), 4 μL of Superscript II first-strand buffer, 1 μl of DTT (100mM), 4 μl of 5 M Betaine, 0.12 μl of 1 M MgCl2, 0.2 μl of TSO (10 μM), 0.58 μl of nuclease-free water) was added to the RNA solution and incubated at 42°C for 90 min, followed by 10 cycles of (50°C for 2 min, 42°C for 2 min), and at 70°C for 15 min. 25 μl KAPA HiFi HotStart ReadyMix, 0.5 μl of 10 μM IS PCR primers, 2.5 μl of 20x Evagreen, and 4 μl of RNase-free water were then added. cDNA was amplified by incubation at 98°C for 1 min, followed by 9-11 cycles (to achieve an increase of 50-100 RFU) of (98°C for 15 s, 67°C for 30 s, 72°C for 6 min). After amplification, cDNA was purified using 0.8× SPRIselect beads. Tn5 transposase (14-17 mg/ml) was produced following a published protocol56.  Tn5 transposase and T5/T7 transposons were assembled to form Tn5 transposome using a published protocol57. Briefly, 1.5 μl of 100 μM T5 transposon (IDT) was mixed with 1.5 μl of 100 μM pMENTS (a 5'-phosphorylated 19-bp mosaic end complementary oligonucleotide, IDT), while 1.5 μl of 100 μM T7 transposon (IDT) was mixed with 1.5 μl of 100 μM pMENTS. 3 μl of T5-pMENTS solution and 3 μl of T7-pMENTS solution were separately incubated at 95℃ for 5 min followed by a slow ramp to 25℃ at -0.1℃/sec. 2.5 μl of T5-pMENTS solution was then mixed with 2.5 μl of Tn5 transposase (14-17 mg/ml), while 2.5 μl of T7-pMENTS solution was mixed with 2.5 μl of Tn5 transposase. T5-Tn5 solution and T7-Tn5 solution were separately incubated at 37℃ for 1 h to form T5-Tn5 transposome and T7-Tn5 transposome, respectively. 4 μl of T5-Tn5 transposome and 4 μl of T7-Tn5 transposome were mixed to form 8 μl of assembled Tn5 transposome before it was mixed with 1 μl cDNA (containing ~500 pg cDNA) and 1 μl reaction buffer (TNP92110, Lucigen). The reaction mixture was incubated at 37 ℃ for 1 h. 1 μl of 10× stop solution (TNP92110, Lucigen) was added to the reaction mixture to stop tagmentation. Two rounds of 1× SPRIselect beads cleanup were performed to purify tagmented cDNA. The tagmented cDNA was then eluted in 10 μl low EDTA TE buffer. 25 μl KAPA HiFi HotStart ReadyMix was activated at 98 ℃ for 30 sec and then mixed with 9.5 μl tagmented cDNA and 10 μl Nuclease-free water, followed by incubation at 72 ℃ for 5 min. 1.5 μl of 25 M P5 primer, 1.5 μl of 25 M P7 primer and 2.5 μl 20x Evagreen were then added. The following amplification program was used to achieve a 300-400 RFU increase: 98 ℃ for 30 s; 11-12 cycles of (98 ℃ for 10 s, 63 ℃ for 30 s, 72 ℃ for 30 s) and 72 ℃ for 1 min. Amplified library was purified with 0.8× SPRIselect beads and eluted in 8 μl low EDTA TE buffer.  Library quality control and sequencing:  Library size and concentration were measured by Tapestation (Agilent) and KAPA Library Quantification Kit (07960140001, Roche), respectively. Libraries were pooled for sequencing by Illumina HiSeq 4000 with single-end 50 nt reads or NovaSeq 6000 in S4 200 mode with paired-end 100 nt reads. Averagely, 19 million reads were generated for each ChIP-seq library, 12 million reads for each ChIP-seq input library, and 16 million reads for each RNA-seq library.
ChIP-seq, RNA-seq


title: M2-Right-10-Neuron-H3K27me3-R1


title: DKO GSK343 Rep1


title: B2M exon1 - mRNA - Rep2

tag: cell type

value: mRNA


title: B2M exon1 - mRNA - Rep1


title: B2M exon1 - LNP - Rep2

value: LNP


title: B2M exon1 - LNP - Rep1


title: DKO DMSO Rep3

value: DMSO


title: CD40L - LNP - Rep3

source_name: T Cells

value: T Cells


title: M2-Right-10-Glia-H3K27me3-R2


title: CD40L - mRNA - Rep3


title: DKO DMSO Rep2


title: B2M exon1 - RNP - Rep1


title: M2-Right-10-Glia-H3K27me3-R1


title: M2-Right-09-Neuron-H3K27me3-R2


title: DKO DMSO Rep1


title: M2-Right-09-Neuron-H3K27me3-R1


title: CD40L - LNP - Rep2


title: CD40L - RNP - Rep3


title: CD40L - RNP - Rep2


title: CD40L - mRNA - Rep2


title: CD40L - mRNA - Rep1


title: CD40L - LNP - Rep1


title: Cgas GSK343 Rep3

value: Cgas KO


title: M2-Right-09-Glia-H3K27me3-R2


title: M2-Right-09-Glia-H3K27me3-R1


title: CD40L - RNP - Rep1


title: Cgas GSK343 Rep1


title: AAVS1 - RNP BM After Transpl. - Rep7

source_name: Bone Marrow

value: Bone Marrow


title: AAVS1 - RNP BM After Transpl. - Rep6


title: Cgas GSK343 Rep2


title: M2-Right-08-Neuron-H3K27me3-R2


title: AAVS1 - RNP BM After Transpl. - Rep5


title: Cgas DMSO Rep3


title: M2-Right-08-Neuron-H3K27me3-R1


title: AAVS1 - RNP BM After Transpl. - Rep4


title: AAVS1 - RNP BM After Transpl. - Rep3


title: M2-Right-08-Glia-H3K27me3-R2


title: Cgas DMSO Rep1


title: Cgas DMSO Rep2


title: AAVS1 - LNPs BM After Transpl. - Rep6


title: AAVS1 - RNP BM After Transpl. - Rep1


title: AAVS1 - LNPs BM After Transpl. - Rep5


title: M2-Right-08-Glia-H3K27me3-R1


title: M2-Right-07-Neuron-H3K27me3-R2


title: AAVS1 - LNPs BM After Transpl. - Rep4


title: AAVS1 - LNPs BM After Transpl. - Rep3


title: M2-Right-07-Neuron-H3K27me3-R1


title: RIG GSK343 Rep2

value: Rigi KO


title: AAVS1 - LNPs BM After Transpl. - Rep2


title: RIG GSK343 Rep3


title: RIG GSK343 Rep1


title: M2-Right-07-Glia-H3K27me3-R2


title: RIG DMSO Rep3


title: AAVS1 - LNPs - Rep3


title: AAVS1 - LNPs - Rep2


title: AAVS1 - LNPs BM After Transpl. - Rep1


title: AAVS1 - mRNA 2 ug - Rep2


title: M2-Right-06-Neuron-H3K27me3-R2


title: M2-Right-07-Glia-H3K27me3-R1


title: AAVS1 - mRNA 2 ug - Rep3


title: M2-Right-06-Neuron-H3K27me3-R1


title: RIG DMSO Rep2


title: AAVS1 - RNP BM After Transpl. - Rep2


title: RIG DMSO Rep1


title: AAVS1 - mRNA 2 ug - Rep1


title: AAVS1 - mRNA 1 ug - Rep2


title: Control GSK343 Rep3

value: WT


title: M2-Right-06-Glia-H3K27me3-R2


title: Control GSK343 Rep2


title: AAVS1 - RNP - Rep2


title: AAVS1 - RNP - Rep3


title: AAVS1 - LNPs - Rep1


title: M2-Right-06-Glia-H3K27me3-R1


title: Control GSK343 Rep1


title: M2-Right-05-Neuron-H3K27me3-R1


title: B2M exon2 - mRNA 1.5 ug T cells - Rep3

value: B2M exon2


title: Control DMSO Rep3


title: M2-Right-05-Neuron-H3K27me3-R2


title: AAVS1 - RNP - Rep1


title: B2M exon2 - RNP 50 pmol T cells - Rep2


title: B2M exon2 - LNPs 1.5 ug T cells - Rep3


title: AAVS1 - mRNA 1 ug - Rep3


title: Control DMSO Rep2


title: B2M exon2 - mRNA 1.5 ug T cells - Rep2


title: M2-Right-05-Glia-H3K27me3-R2


title: Control DMSO Rep1


title: M2-Right-05-Glia-H3K27me3-R1


title: B2M exon2 - LNPs 1.5 ug T cells - Rep2


title: M2-Right-04-Neuron-H3K27me3-R2


title: B2M exon2 - mRNA 1.5 ug T cells - Rep1


title: B2M exon2 - RNP 50 pmol T cells - Rep1


title: AAVS1 - mRNA 1 ug - Rep1


title: Mutant B cells H3K27me3 ChIP GSK343 Rep2

data_processing:
Base calls and demultiplexing were performed using bcl2fastq
Paired reads were aligned to mm10 using bowtie2/2.4.2 using --sensitive-local
Concordantly mapped reads were obtained from all mapped reads using samtools/1.12 -f 0x2
Broad peaks were called using macs2/2.2.7.1 using -f BAMPE --broad and pooling biological duplicates together
Called broad peaks were then filtered to remove mm10 blacklist regions (ENCODE) using bedtools/2.29.2 intersect -v
bedtools/2.29.2 intersect -v OR -u was used to identify unique or common peaks in pairwise comparisons, respectively
Coverage tracks (excluding any blacklist regions) were generated for each biological sample using bamCoverage (deeptools/3.5.1) --normalizeUsing RPGC -e -bl $/path/to/blacklist/file. Csaw/1.34 R package was used to calculate normalization values for ChIP libraries, which were used as input values for --scaleFactor option.
Data was visualized using deeptools/3.5.1 and IGV/2
Assembly: mm10
Supplementary files format and content: bw: RPGC coverage file from bamCoverage; broadPeak: peak calls from macs2

source_name: Splenic B cells

growth_protocol: Splenic B cells were purified from 6-8 week old mice using MACS and CD43 microbeads (Miltenyi biotec). The cells were treated with either DMSO or  1 uM GSK343 (5x10^6 cells/mL) for 48 hours in RPMI (Wisent) supplemented with 10% (v/v) FBS, 100 IU penicillin, 100 ug/mL streptomycin, 2 mM glutamine,  55 uM 2-BME, 2ng/mL BAFF and 2ng/mL IL-4 (BioLegend).

extract_protocol:
Harvested cells were washed with PBS and fixed with 1% formaldehyde/PBS for 5 min at RT. The reaction was quenched with 0.125 M glycine for 5 min at RT. Fixed cells were washed with PBS and lysed to isolate chromatin. Chromatin was sonicated with Bioruptor Pico (Diagenode) to < 500 bp. Sonicated chromatin was diluted ten-fold and pre-cleared with Dynabeads Protein G (ThermoFisher). A small volume (5%) was saved as input. The remainder was incubated with 4 ug anti-H3K27me3 antibody per 30 ug chromatin over night. Dynabeads Protein G was added and incubated for 2 hours. Immunoprecipitated chromatin-protein complexes were washed and decrosslinked over night at 65 C. Input and ChIP DNA were treated with RNaseA and proteinase K, column purified (NEB T1030S), and quantified with Qubit (ThermoFisher).
Libraries were prepared with NEBNext Ultra II DNA kit (E7645S) and NEBNext Multiplex Oligos (E7600S), following the manufacturer's recommendations. Size selection and purification were performed with Ampure XP beads (Beckman Coulter).

treatment_protocol: DMSO or 1 uM GSK343 for 48 hours

value: MACS-purified splenic B cells

value: Ifih1/Rigi/cGAS triple mutant

value: 1 uM GSK343


title: B2M exon2 - RNP 50 pmol T cells - Rep3


title: M2-Right-04-Neuron-H3K27me3-R1

source_name: C57BL/6J brain

value: brain


title: B2M exon2 - RNP 50 pmol - Rep7

value: Cutured HSPC

value: RNP


title: Mutant B cells H3K27me3 input GSK343 Rep1


title: M2-Right-04-Glia-H3K27me3-R2


title: B2M exon2 - LNPs 1.5 ug - Rep9


title: B2M exon2 - LNPs 1.5 ug - Rep8


title: M2-Right-04-Glia-H3K27me3-R1


title: B2M exon2 - RNP BM After Transpl. - Rep11


title: B2M exon2 - RNP SPL After Transpl. - Rep11

source_name: Spleen

value: Spleen


title: Mutant B cells H3K27me3 input DMSO Rep2


title: M2-Right-03-Neuron-H3K27me3-R2


title: B2M exon2 - RNP PB After Transpl. - Rep11

source_name: Peripheral Blood

value: Peripheral Blood


title: Mutant B cells H3K27me3 ChIP GSK343 Rep1


title: B2M exon2 - RNP BM After Transpl. - Rep10


title: B2M exon2 - RNP SPL After Transpl. - Rep10


title: Mutant B cells H3K27me3 input DMSO Rep1


title: B2M exon2 - RNP SPL After Transpl. - Rep9


title: M2-Right-03-Neuron-H3K27me3-R1


title: B2M exon2 - LNPs 1.5 ug T cells - Rep1


title: B2M exon2 - RNP 50 pmol - Rep8


title: B2M exon2 - RNP BM After Transpl. - Rep9


title: M2-Right-03-Glia-H3K27me3-R1


title: B2M exon2 - RNP PB After Transpl. - Rep9


title: Mutant B cells H3K27me3 ChIP DMSO Rep1


title: B2M exon2 - LNPs 1.5 ug - Rep7


title: B2M exon2 - RNP SPL After Transpl. - Rep8


title: Mutant B cells H3K27me3 input GSK343 Rep2


title: M2-Right-02-Neuron-H3K27me3-R2


title: B2M exon2 - RNP BM After Transpl. - Rep8


title: B2M exon2 - RNP PB After Transpl. - Rep8


title: B2M exon2 - RNP SPL After Transpl. - Rep7


title: Mutant B cells H3K27me3 ChIP DMSO Rep2


title: B2M exon2 - RNP PB After Transpl. - Rep10


title: M2-Right-03-Glia-H3K27me3-R2


title: B2M exon2 - RNP BM After Transpl. - Rep7


title: B2M exon2 - RNP SPL After Transpl. - Rep6


title: WT B cells H3K27me3 input GSK343 Rep1


title: B2M exon2 - RNP BM After Transpl. - Rep6


title: WT B cells H3K27me3 input DMSO Rep2


title: B2M exon2 - RNP PB After Transpl. - Rep6


title: M2-Right-02-Glia-H3K27me3-R1


title: B2M exon2 - LNPs SPL After Transpl. - Rep13


title: M2-Right-01-Neuron-H3K27me3-R2


title: WT B cells H3K27me3 input GSK343 Rep2


title: WT B cells H3K27me3 ChIP DMSO Rep2


title: WT B cells H3K27me3 ChIP GSK343 Rep2


title: WT B cells H3K27me3 input DMSO Rep1


title: B2M exon2 - LNPs PB After Transpl. - Rep13


title: M2-Right-02-Neuron-H3K27me3-R1


title: M2-Right-01-Neuron-H3K27me3-R1


title: WT B cells H3K27me3 ChIP DMSO Rep1


title: B2M exon2 - LNPs BM After Transpl. - Rep12


title: M2-Right-02-Glia-H3K27me3-R2


title: B2M exon2 - LNPs PB After Transpl. - Rep12


title: B2M exon2 - RNP PB After Transpl. - Rep7


title: WT B cells H3K27me3 ChIP GSK343 Rep1


title: B2M exon2 - LNPs BM After Transpl. - Rep11


title: Mutant-B-GSK343 Rep3

data_processing:
Base calls and demultiplexing were performed using bcl2fastq
Gene expression analysis: Reads were mapped to mm10 with STAR/2.7.8a and GENCODE VM22 comprehensive gene annotation. SAM files were converted to BAM, sorted and indexed with samtools/1.12. Reads aligned to gene annotations were counted with htseq-count/0.11.0 using default settings. Count tables were imported into Rstudio using R/3.6.3. Differential gene expression analysis was performed with edgeR/3.28.1.
Repetitive element expression analysis version 1: Reads were mapped to mm10 with bowtie2/2.3.3.1 using default settings. Unique and multimapped reads were separated using RepEnrich2. RepEnrich2 was used to count the number of reads aligning to each repName element in a custom repeat annotation file. The annotation was generated by removing low complexity and simple repeat repClass elements from Repeat Masker table downloaded from UCSC Table Browser. Any element overlapping mm10 blacklist coordinates (ENCODE) was also removed. Count tables were imported into Rstudio using R/3.6.3. Differential gene expression analysis was performed with edgeR/3.28.1.
Repetitive element expression analysis version 2: Reads were mapped to mm10 with STAR/2.5.2b using previously described settings (see citation in paper). Mapped reads overlapping with features in RepeatMasker track (mm10) were counted using FeatureCounts/2.0.3. Count tables were imported into Rstudio using R/3.6.3. Differential gene expression analysis was performed with edgeR/3.28.1.
Assembly: mm10
Supplementary files format and content: csv: count table of gene expression; txt: count table of repetitive element expression (Repenrich2); table: count table of repetitive element expression (STAR and featureCounts)

growth_protocol: Splenic B cells were purified from 6-8 week old mice using MACS and CD43 microbeads (Miltenyi biotec). The cells were treated with either DMSO or  0.5 uM GSK343 (5x10^6 cells/mL) for 48 hours in RPMI (Wisent) supplemented with 10% (v/v) FBS, 100 IU penicillin, 100 ug/mL streptomycin, 2 mM glutamine,  55 uM 2-BME, 2ng/mL BAX and 2ng/mL IL-4 (BioLegend).

treatment_protocol: DMSO or 0.5 uM GSK343 for 48 hours

value: Ifih1/Ddx58/cGAS triple mutant


title: B2M exon2 - LNPs PB After Transpl. - Rep11

value: LNP


title: M2-Left-20-Neuron-H3K27me3-R2


title: M2-Right-01-Glia-H3K27me3-R2


title: M2-Right-01-Glia-H3K27me3-R1


title: B2M exon2 - LNPs SPL After Transpl. - Rep10


title: M2-Left-20-Neuron-H3K27me3-R1


title: B2M exon2 - LNPs BM After Transpl. - Rep10


title: B2M exon2 - LNPs BM After Transpl. - Rep13


title: B2M exon2 - LNPs PB After Transpl. - Rep10


title: Mutant-B-GSK343 Rep1


title: B2M exon2 - LNPs SPL After Transpl. - Rep12


title: B2M exon2 - LNPs SPL After Transpl. - Rep9


title: Mutant-B-GSK343 Rep2


title: M2-Left-20-Glia-H3K27me3-R2


title: B2M exon2 - LNPs BM After Transpl. - Rep9


title: B2M exon2 - LNPs SPL After Transpl. - Rep11


title: Mutant-B-DMSO Rep3

value: DMSO


title: B2M exon2 - LNPs PB After Transpl. - Rep9


title: M2-Left-19-Neuron-H3K27me3-R2


title: M2-Left-20-Glia-H3K27me3-R1


title: B2M exon2 - LNPs SPL After Transpl. - Rep8


title: B2M exon2 - LNPs BM After Transpl. - Rep8


title: B2M exon2 - LNPs PB After Transpl. - Rep8


title: B2M exon2 - LNPs SPL After Transpl. - Rep7


title: Mutant-B-DMSO Rep1


title: M2-Left-19-Glia-H3K27me3-R2


title: WT-B-GSK343 Rep2


title: M2-Left-19-Glia-H3K27me3-R1


title: B2M exon2 - RNP SPL After Transpl. - Rep5


title: WT-B-GSK343 Rep3


title: B2M exon2 - LNPs BM After Transpl. - Rep7


title: M2-Left-18-Neuron-H3K27me3-R2


title: B2M exon2 - RNP PB After Transpl. - Rep5


title: B2M exon2 - RNP SPL After Transpl. - Rep4


title: B2M exon2 - RNP BM After Transpl. - Rep4


title: WT-B-DMSO Rep3


title: M2-Left-18-Neuron-H3K27me3-R1


title: WT-B-GSK343 Rep1


title: B2M exon2 - RNP PB After Transpl. - Rep4


title: M2-Left-18-Glia-H3K27me3-R2


title: M2-Left-19-Neuron-H3K27me3-R1


title: B2M exon2 - RNP BM After Transpl. - Rep3


title: WT-B-DMSO Rep2


title: B2M exon2 - RNP PB After Transpl. - Rep3


title: B2M exon2 - RNP SPL After Transpl. - Rep3


title: WT-B-DMSO Rep1


title: B2M exon2 - RNP PB After Transpl. - Rep2


title: B2M exon2 - RNP BM After Transpl. - Rep2


title: B2M exon2 - RNP SPL After Transpl. - Rep2


title: Splenocyte-GSK343 Rep2

source_name: Splenic lymphocytes

value: RBC-lysed splenic lymphocytes


title: M2-Left-17-Neuron-H3K27me3-R2


title: B2M exon2 - RNP SPL After Transpl. - Rep1


title: M2-Left-17-Neuron-H3K27me3-R1


title: B2M exon2 - RNP BM After Transpl. - Rep5


title: B2M exon2 - RNP PB After Transpl. - Rep1


title: M2-Left-17-Glia-H3K27me3-R2


title: B2M exon2 - mRNA SPL After Transpl. - Rep5

value: mRNA


title: Splenocyte-GSK343 Rep1


title: M2-Left-18-Glia-H3K27me3-R1


title: B2M exon2 - mRNA BM After Transpl. - Rep5

value: Bone Marrow


title: Splenocyte-GSK343 Rep3


title: M2-Left-17-Glia-H3K27me3-R1


title: Splenocyte-DMSO Rep2


title: B2M exon2 - mRNA PB After Transpl. - Rep4


title: M2-Left-16-Neuron-H3K27me3-R1


title: Splenocyte-DMSO Rep1


title: B2M exon2 - mRNA SPL After Transpl. - Rep4


title: B2M exon2 - LNPs PB After Transpl. - Rep7


title: Mutant-B-DMSO Rep2


title: M2-Left-16-Glia-H3K27me3-R1


title: M2-Left-16-Glia-H3K27me3-R2


title: Pilot_inVivo_test_Cas9_rep2

city: Hangzhou

first: SHOU-WEN

last: WANG

state: Zhejiang Province

address: Shilongshan ST #18, Xihu District

institute: Westlake University

data_processing: DATA are processed with CARLIN pipeline, except CARLIN libraries from Camellia-seq, which were analyzied with a customized script.

source_name: liver

extract_protocol:
Each sample has its corresponding CARLIN protocol as shown above. The CARLIN DNA or RNA materials are extracted from either 1) bulk cell populations sorted with FACS or 2) single-cell samples prepared with 10X encapsulation or individually sorted single cells from Camellia-seq
We used bulk RNA or DNA as starting material. RNA were converted to DNA with SuperScrit III. Then we used a nested PCR approach with locus specific primers to perform targeted amplification with Q5 High-Fidelity DNA Polymerase (New England Biosciences, M0491L). We used 1.5X Ampure XP beads (Beckman Coulter, A63881) to purify the PCR product from each step once except the final indexing PCR product (0.8X, twice purification). The final indexing libraries were pooled and quantified (Kappa Biosystems, KK4835) and sequenced on Illumina MiSeq using paired-end 500 cycles v2 kits (Illumina, MS-102-2003) with 5% PhiX sequencing control v3 (Illumina, FC-110-3001).

value: liver

tag: carlin protocol

tag: carlin locus

value: CC


title: B2M exon2 - mRNA PB After Transpl. - Rep3

value: B2M exon2


title: M2-Left-15-Neuron-H3K27me3-R2

tag: strain


title: B2M exon2 - mRNA BM After Transpl. - Rep4

tag: treatment


title: M2-Left-16-Neuron-H3K27me3-R2


title: Pilot_inVivo_test_Cas9-TdT_rep2


title: B2M exon2 - mRNA SPL After Transpl. - Rep3


title: Pilot_inVivo_test_Cas9-TdT_rep1


title: ES_time_course_DNA-96h-MediumDox

source_name: ESC

value: ESC

value: TC


title: M2-Left-15-Glia-H3K27me3-R2


title: ES_time_course_DNA-96h-LowDox


title: M2-Left-15-Glia-H3K27me3-R1


title: B2M exon2 - mRNA SPL After Transpl. - Rep2


title: Sample 9_CD45+ immune cells and CD45- non-immune cells, NDV-GP, scTCR-seq

first: Ziyu

last: Li

address: 5 Summer Palace Road, Haidian District, Beijing, China

department: Biomedical Pioneering Innovation Center

institute: Peking University

description: 10x Genomics (scTCR-seq)

data_processing:
library strategy: scTCR-seq
The demultiplexing, barcoded processing, gene counting were made using the Cell Ranger software v6.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/What-is-cell-ranger)
Assembly: mm10

molecule: polyA RNA

source_name: MC38 tumor

extract_protocol: For sample 1-3, total RNA of each sample was harvested using  Micro Total RNA Isolation Kit (Invitrogen, AM1931). For sample 4-9, RNA extraction of each sample was included in the 10X genomics scRNA-seq/scTCR-seq kit.

value: MC38 tumor

tag: cell line

value: Tumor-infiltrating CD45+ and CD45- cells

value: WT

value: NDV-GP


title: ES_time_course_DNA-96h-HighDox


title: Sample 8_CD45+ immune cells and CD45- non-immune cells, NDV-WT, scTCR-seq

description: 10x Genomics (scTCR-seq)

tag: genotype

value: NDV-WT


title: M2-Left-14-Neuron-H3K27me3-R2

value: brain


title: B2M exon2 - mRNA BM After Transpl. - Rep2


title: Sample 7_CD45+ immune cells and CD45- non-immune cells, PBS, scTCR-seq

description: 10x Genomics (scTCR-seq)

value: PBS


title: Pilot_inVivo_test_Cas9_rep1


title: M2-Left-14-Neuron-H3K27me3-R1


title: ES_time_course_DNA-96h-0Dox


title: M2-Left-15-Neuron-H3K27me3-R1


title: Sample 6_CD45+ immune cells and CD45- non-immune cells, NDV-GP, scRNA-seq

description: 10x Genomics (scRNA-seq)

data_processing:
The demultiplexing, barcoded processing, gene counting were made using the Cell Ranger software v6.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/What-is-cell-ranger)
Assembly: mm10


title: B2M exon2 - mRNA SPL After Transpl. - Rep1

value: Spleen


title: M2-Left-14-Glia-H3K27me3-R2


title: Sample 5_CD45+ immune cells and CD45- non-immune cells, NDV-WT, scRNA-seq

description: 10x Genomics (scRNA-seq)


title: ES_time_course_DNA-72h-MediumDox


title: B2M exon2 - mRNA BM After Transpl. - Rep3


title: ES_time_course_DNA-72h-LowDox


title: B2M exon2 - mRNA BM After Transpl. - Rep1


title: M2-Left-14-Glia-H3K27me3-R1


title: Sample 4_CD45+ immune cells and CD45- non-immune cells, PBS, scRNA-seq

description: 10x Genomics (scRNA-seq)


title: M2-Left-13-Neuron-H3K27me3-R2


title: ES_time_course_DNA-72h-HighDox


title: ES_time_course_DNA-72h-0Dox


title: Sample 3_p15E+CD8+ T cell, NDV-GP, TCR-seq

data_processing:
library strategy: TCR-seq
The demultiplexing, barcoded processing, gene counting were made using the Cell Ranger software v6.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/What-is-cell-ranger)
Assembly: mm10

value: Immune cell

value: CD8+ T cell


title: M2-Left-13-Neuron-H3K27me3-R1


title: B2M exon2 - LNPs SPL After Transpl. - Rep6

value: LNP


title: ES_time_course_DNA-48h-MediumDox


title: M2-Left-13-Glia-H3K27me3-R2


title: Sample 2_p15E+CD8+ T cell, NDV-WT, TCR-seq


title: B2M exon2 - LNPs BM After Transpl. - Rep6

value: Bone Marrow


title: ES_time_course_DNA-48h-LowDox


title: M2-Left-13-Glia-H3K27me3-R1


title: B2M exon2 - mRNA PB After Transpl. - Rep2

value: Peripheral Blood

value: mRNA


title: B2M exon2 - LNPs PB After Transpl. - Rep6

value: B2M exon2


title: M2-Left-12-Neuron-H3K27me3-R2


title: DNA mutant WGS-seq

city: Caserta

first: Francesco

last: Cecere

state: Caserta

address: Via A. Vivaldi, n. 43

department: DiStABiF

institute: University of Campania "Luigi Vanvitelli"

data_processing:
The read quality was checked using the FASTQC tools (Andrews S, 2010). Reads were aligned to the mouse genome reference assembly (mm39) using the BWAmem software package v0.7.17 (Li H, 2009). PCR duplicates were filtered out by Picard v2.9 (http://picard.source forge.net), and the Dellytools v1.1.6 with default parameters were employed to calculate the structural and CNV variants.
Assembly: mm39
Supplementary files format and content: VCF file for Svs

source_name: Tail

growth_protocol: For the experiment, males of 8-12 weeks of age and females of 4-8 weeks of age were used. The day before the start of the experiment, the males were separated. On day 1, at 2 pm, intraperitoneal injections of pregnant mare serum gonadotropin (PMSG) were given to the females. After approximately 52 hours, the same females received intraperitoneal injections of human chorionic gonadotropin (hCG). Male was sacrificed approximately 12 hours after the female hCG injection. The cauda epididymis and vasa deferentia were dissected and the spermatozoa were collected in the human tubal fluid medium (HTF) (EmbryoMax® HTF MR-070-D). Spermatozoa were counted and incubated at 37°C within a humidified atmosphere of 5% CO2 in air. Females were sacrificed approximately 13 hours after the hCG injection. Ampullae were placed in hyaluronidase 1mg/ml (H6254-500MG) drops to break down the oocyte-cumulus complex and collect MII oocyte. The oocytes were moved to the HTF medium and a 2x106 sperm/ml concentration of spermatozoa were added. After 4 - 6 hours of incubation at 37°C within a humidified atmosphere of 5% CO2 in air, the fertilised oocytes were washed, to remove the spermatozoa e debris, transferred in KSOM medium and incubated overnight at 37°C within a humidified atmosphere of 5% CO2 in air. The embryos' status was evaluated in the morning and the next 120 hours.

extract_protocol:
The presence of the Padi6 variant, the removal of Neo box and the presence of the flippase gene were demonstrated by PCR on tail DNA extracted with standard procedures. Amplicons of 430 bp and 290 bp were amplified from the mutant or wild type Padi6 alleles, respectively, by using the primers Padi6_RimF and Padi6_RimR. This difference was due to a fragment of the NEO box remaining after the flippase recombination. The presence of the mutation was also identified by digestion with the restriction enzyme StuI, which was generated by the mutation, after amplification of a 420 bp fragment with the primers For LA HindIII + SnaBI and Rev GEN Padi6. The presence of the NEO box was detected using the primers PGK1A For and Padi6_RimR. The presence of the flippase was detected using the primers FLP_F and FLP_R. The primer sequences are listed in the Supp. Table S17.
Whole genome sequencing was performed on genomic DNA of homozygous female. The DNA samples were sequenced 150 bp pair-end at BIODIVERSA srl Service (Milan, Italy) using the Nextera DNA Prep illumina and the Illumina NovaSeq6000 platform.

value: Tail


title: B2M exon2 - LNPs SPL After Transpl. - Rep5


title: B2M exon2 - mRNA PB After Transpl. - Rep1


title: ES_time_course_DNA-48h-HighDox

value: ESC

value: TC


title: KO_Mouse_Islet4

city: San Francisco

first: Sapna

last: Puri

address: 513 Parnassus Avenue

institute: UCSF

data_processing:
mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The mRNA was fragmented randomly by addition of fragmentation buffer. Then first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library was gotten by PCR amplification and purification of PCR products by AMPure XP beads.
After library construction, diluting library to 1.5ng/ul with the preliminary quantitative result by Qubit2.0 and detecting the insert size by Agilent 2100. Q-PCR is used to accurately quantify the library effective concentration (> 2nM), in order to ensure the library quality. Libraries are fed into illumina machines after pooling according to activity and expected data volume if library quality is up to standard.
Raw image data file from high-throughput sequencing(like Illumina ) was transformed to Sequenced Reads(called Raw Data or Raw Reads) by CASAVA base recognition (Base Calling). Raw Data is stored in FASTQ(fq) format files, which contain reads sequence and corresponding base quality. STAR software was used for alignment for RNA-seq sequencing data analysis.
The raw fastq files were adapter-trimmed with cutadapt v1.13 and were subsequently processed with kallisto version 0.46.1 using the Ensembl GRCm38 as the reference transcriptome for building the index. The kallisto quantification was bootstrapped 100 times (using the -b parameter) and the output was processed for differential expression analysis using the sleuth package in R. We first performed a gene-level Wald test (gene_mode=True) between Sox9 knockout (KO) and control (WT) by aggregating the estimated isoform counts of each gene to their corresponding ensembl gene id in each condition.
In order to further capture alternative splicing and isoform switching events we performed a transcript-level Wald test and aggregated the resulting p-values to the gene-level (aggregation_column=’ens_gene’). This approach uses the Lancaster method to compute a single p-value for each gene taking into account the individual p-values of its isoforms. In both gene- and transcript- level analyses, the raw p-values were used to compute Benjamini-Hochberg-adjusted false discovery rates (q values) and significance was set at 5% FDR throughout the manuscript.
Assembly: mm10
Supplementary files format and content: Excel File containing the reads for each biological replicate for both groups.

source_name: Pancreatic Islets

growth_protocol: Primary tissue (islets) isolated from mice

extract_protocol:
Total RNA was extracted from isolated islets using the Rneasy Mini kit from Qiagen as per manufacturer's instructions.
RNA-seq libraries were prepared using the KAPA Stranded RNA-Seq Kits as per the manufacturer's instructions.

treatment_protocol: Genetic knockout animal models

value: Pancreatic Islets

value: Mixed

tag: age

value: 13-16 months old mice

value: Islet cells


title: B2M exon2 - LNPs BM After Transpl. - Rep5


title: B2M exon2 - LNPs PB After Transpl. - Rep5


title: M2-Left-12-Neuron-H3K27me3-R1

value: brain


title: ES_time_course_DNA-24h-MediumDox