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


title: KO_Mouse_Islet2

status: Public on Oct 18 2023

type: SRA

city: San Francisco

first: Sapna

last: Puri

address: 513 Parnassus Avenue

country: USA

institute: UCSF

library_source: transcriptomic

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.

molecule: total RNA

organism: Mus musculus

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

tag: tissue

value: Pancreatic Islets

tag: strain

value: Mixed

tag: age

value: 13-16 months old mice

tag: cell type

value: Islet cells

tag: genotype


title: ES_time_course_DNA-48h-0Dox

city: Hangzhou

first: SHOU-WEN

last: WANG

state: Zhejiang Province

address: Shilongshan ST #18, Xihu District

country: China

institute: Westlake University

library_source: genomic

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

molecule: genomic DNA

source_name: ESC

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: ESC

tag: carlin protocol

tag: carlin locus

value: TC


title: ES_time_course_DNA-24h-LowDox


title: KO_Mouse_Islet3


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

city: Blacksburg

first: Chang

last: Lu

state: VA

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

department: Chemical Engineering

institute: Virginia Tech

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

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


title: ES_time_course_DNA-24h-HighDox


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


title: KO_Mouse_Islet1


title: WT_Mouse_Islet4


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


title: Sample 1_p15E+CD8+ T cell, PBS, TCR-seq

city: Beijing

first: Ziyu

last: Li

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

department: Biomedical Pioneering Innovation Center

institute: Peking University

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

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: Immune cell

value: CD8+ T cell

value: WT

tag: treatment

value: PBS


title: ES_time_course_DNA-24h-0Dox


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

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

organism: Homo sapiens

source_name: Peripheral Blood

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: Peripheral Blood

tag: target locus

value: B2M exon2

value: LNP


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

source_name: Bone Marrow

value: Bone Marrow


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

source_name: Spleen

value: Spleen


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


title: ES_time_course_DNA-12h-LowDox


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


title: WT_Mouse_Islet2

value: Pancreatic Islets


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


title: ES_time_course_DNA-12h-HighDox


title: ES_time_course_DNA-12h-0Dox


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


title: WT_Mouse_Islet1

value: Mixed


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


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


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


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


title: WT TCD2  (G3_2)

city: Brisbane

first: Rachael

last: Adams

state: QLD

address: 300 Herston Road

country: Australia

institute: QIMR Berghofer Medical Research Institute

description: 10X Genomics

library_source: transcriptomic single cell

data_processing:
Demultiplexing, alignment to the Mus musculus genome, filtering, barcode counting, and UMI counting were performed using CellRanger v6.0.1.
Assembly: Mus musculus GRCm39
Supplementary files format and content: RDS file
Supplementary files format and content: Matrix files (filtered deature-barcode matrix)

source_name: Brain

extract_protocol:
On day 70 post-transplant, mice were transcardially perfused with 1X PBS and the brain dissected. Brain tissue was weighed to obtain a 50mg sample, washed with cold 1X PBS, and flash-frozen in liquid nitrogen. Nuclei isolation was performed using the 10X Genomics Chromium Nuclei Isolation Kit as per the manufacturer's instructions. THe nuclei product was stained with DAPI and sort purified for DAPI+ nuclei. Nuclei were immediately processed for sequencing.
The library was constructed as per the manufacturer's instructions (10X Genomics, Chromium Next GEM Single Cell 3' v3.1). Briefly, nuclei were resuspended in the mastermix and loaded into the Chromium Chip, along with gel beads and partioning oil, to form the GEMs. Gel beads were dissolved, and primers were released, containing Illumina TruSeq Read 1 sequencing primers, 16nt 10x barcode, 12nt Unique Molecular Identifier, and 30nt poly(dT) sequence. Reverse transcription was used to produce barcoded, full-length cNDA from the poly-adenylated mRNA. The cDNA was then purified with magnetic beads and amplified via PCR to generate required amounts for library construction. SPRISelect was used to perform size selection. P5, P7, TruSeq Read 2 sequencing primers and a sample index were added during library construction.

value: Brain

tag: Sex

value: Female

value: 12 weeks old

value: B6.Csf1r-eGFP x DBA2 F1

value: Allogeneic transplantion, no donor T cells


title: WT TCD1  (G3_1)

description: 10X Genomics


title: WT_Mouse_Islet3

value: 13-16 months old mice

value: Islet cells


title: ES_time_course_DNA-0h-0dox

value: ESC

value: TC


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

value: brain


title: DARLIN_v1_tissues_LL211-spleen

source_name: spleen

value: spleen

value: CC


title: ES_time_course_DNA-12h-MediumDox


title: IFNGRKO GVHD1 (G2_1)

description: 10X Genomics

value: Allogeneic transplantion, IFNGR knockout bone marrow and WT donor T cells


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


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

value: Bone Marrow

value: B2M exon2

value: LNP


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


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

value: Peripheral Blood


title: DARLIN_v1_tissues_LL211-ovary

source_name: ovary

value: ovary


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

value: RNP


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


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


title: IFNGRKO GVHD2 (G2_2)

description: 10X Genomics

value: Brain

value: Female

value: 12 weeks old


title: DARLIN_v1_tissues_LL211-liver

source_name: liver

value: liver


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


title: WT GVHD1 (G1_1)

description: 10X Genomics

value: B6.Csf1r-eGFP x DBA2 F1

value: Allogeneic transplantion, WT donor bone marrow and T cells


title: WT GVHD2 (G1_2)

description: 10X Genomics


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


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

value: Spleen


title: DARLIN_v1_tissues_LL211-kidney

source_name: kidney

value: kidney


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

value: brain


title: DARLIN_v1_tissues_LL211-intestine

source_name: intestine

value: intestine

value: CC


title: B2M exon2 - UT - Rep6

source_name: Cutured HSPC

value: Cutured HSPC

value: Untreated


title: mK4 cells

city: Chongqing

first: Lei

address: yixueyuan road

institute: CQMU

data_processing:
For RNA-seq, the STAR software (version 2.7.10a) was used to build the reference genome index and perform the alignment of paired-end clean reads to the reference genome under the Ubuntu environment. For ATAC-seq, The mouse genome reference sequence mm10 was used for bowtie2-based alignment of the raw data.
For RNA-seq, HTSeq software (version 2.0.2)  for Ubuntu was employed to calculate gene counts and FPKM values (Fragments Per Kilobase of transcript sequence per Millions of base pairs sequenced) from the aligned transcriptomic sequencing BAM files and the reference genome annotation GTF file for gene expression quantification.
For ATAC-seq, PCR duplicates were removed using the Sambamba software. Low-quality alignments and mitochondrial sequences were filtered out from the *_rdup.bam files using samtools and grep commands to produce *_rdup_rMT.bam files, which were then indexed. Samtools [90] was used to characterize the sequencing depth, coverage, alignment rate, and duplication rate of the processed *_rdup_rMT.bam files.
For ATAC-seq, we used Bedtools to convert the *_rdup_rMT.bam files to .bed files for subsequent analyses. Insert size calculations and peak calling were performed using Bedtools intersect and MACS3 software (github.com/macs3-project/MACS), respectively, to identify open chromatin regions.
For ATAC-seq, DeepTools was used to generate bigwig files for visualization, and computeMatrix, plotHeatmap, and plotProfile were employed to visualize the ATAC-seq signal.
Assembly: mm10
Supplementary files format and content: (RNA-seq) tab-delimited text file includes raw counts for each sample
Supplementary files format and content: (ATAC-seq) bigWig

growth_protocol: Both mK3 and mK4 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in humidified atmosphere with 5% CO2 at 37℃

extract_protocol:
Total RNA was extracted from mouse kidney cells using TRIzol reagent (Invitrogen) according to manual instructions
For RNA-seq, Novogene employs a ribosomal RNA depletion method (with an additional linear RNA removal step for circRNA library construction) to build strand-specific libraries. Initially, ribosomal RNA is removed from total RNA, followed by fragmentation of the RNA into 250-300 bp segments. These fragmented RNAs serve as templates for the first strand cDNA synthesis, initiated by random primers. The second cDNA strand is then synthesized using dNTPs (including dUTP, dATP, dGTP, and dCTP). The resulting double-stranded cDNA is purified, end-repaired, adenylated, and ligated with sequencing adapters. Fragments around 350-400 bp are selected using AMPure XP beads. USER enzyme is employed to digest the second cDNA strand containing 'U', followed by PCR amplification to obtain the final library. Strand-specific libraries offer numerous advantages such as acquiring more valuable information at the same data volume and achieving precise gene quantification, localization, and annotation. For ATAC-seq, upon sample verification, a transposase mix containing the transposase and equimolar adapters Adapter 1 and Adapter 2 is added. Incubation at 37°C for 30 minutes facilitates transposition and simultaneous adapter ligation at the fragment ends. These fragmented products, post-primer amplification, fragment length selection, and purification, constitute the final sequencing library.

value: epithelial-differentiated metanephric mesenchymal cells

value: wt


title: DARLIN_v1_tissues_LL211-lung

source_name: lung

value: lung


title: mK3 cells

value: uninduced metanephric mesenchymal cells


title: DARLIN_v1_tissues_LL196-testis

source_name: testis

value: testis


title: B2M exon2 - UT - Rep4

value: B2M exon2


title: mK4 cells, dup #3


title: B2M exon2 - UT - Rep5


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


title: DARLIN_v1_tissues_LL196-spleen

value: spleen


title: mK4 cells, dup #2


title: B2M exon2 - UT - Rep3


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


title: mK4 cells, dup #1


title: DARLIN_v1_tissues_LL196-lung


title: B2M exon2 - UT - Rep2


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


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


title: mK3 cells, dup #3


title: B2M exon2 - UT - Rep1


title: DARLIN_v1_tissues_LL196-liver

value: liver


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


title: DARLIN_v1_tissues_LL196-kidney


title: B2M exon2 - RNP 25 pmol - Rep6

value: RNP


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


title: DARLIN_v1_tissues_LL195-spleen


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


title: mK3 cells, dup #1


title: DARLIN_v1_tissues_LL196-intestine


title: B2M exon2 - RNP 25 pmol - Rep4


title: B2M exon2 - RNP 25 pmol - Rep5


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


title: DARLIN_v1_tissues_LL195-ovary

value: ovary


title: lps_dmso_2

city: Gainesville

first: Jake

last: Boles

state: FL

address: LG-152, 1149 Newell Dr

department: Neuroscience

institute: University of Florida

data_processing:
FASTQ quality control, demultiplexing, barcode processing, and gene mapping/counting were accomplished with the PIPseeker software v.2 (https://www.fluentbio.com/products/pipseeker-software-for-data-analysis/)
Cell calling, clustering, and cleaning was performed in R with the Seurat package v.4.
All downstream analysis of the processed data files included can be found at https://github.com/jakesboles/Boles_et-al_brain_immune_scRNAseq
Assembly: GRCmm39
Supplementary files format and content: Tab-separated and matrix files from PIPseeker.

source_name: brain

extract_protocol:
40,000 cells per sample were input into the PIPseq protocol to capture ~20,000 cells per sample. Cells were captured in PIPs and lysed, enabling RNA to be captured and barcoded, as per manufacturer's instructions.
Single-cell cDNA libraries were prepared using the T20 PIPseq kit, adhering to manufacturer's instructions.

treatment_protocol: Mice received 1.5e7 EU/kg of LPS or an equivalent volume of sterile saline via single intraperitoneal injection. Mice were euthanized via rapid decapitation and brains were extracted 24 hours later and dissociated using the Adult Brain Dissociation Kit with the octoMACS. CD45+ cells were isolated from brains using magnetic separation.

value: CD45+ cells

tag: in vivo_treatment

value: LPS

tag: ex vivo_treatment

value: DMSO

tag: batch

value: 5-6 months old

value: Male


title: lps_inhibitors_2

value: brain

value: Inhibitors


title: saline_inhibitors_2

value: Saline


title: B2M exon2 - RNP 25 pmol - Rep2

value: Cutured HSPC


title: B2M exon2 - RNP 25 pmol - Rep1


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


title: DARLIN_v1_tissues_LL195-lung

value: lung

value: CC


title: Splenocyte-DMSO 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 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)

source_name: Splenic lymphocytes

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).

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 0.5 uM GSK343 for 48 hours

value: WT

value: RBC-lysed splenic lymphocytes


title: mK3 cells, dup #2

value: uninduced metanephric mesenchymal cells

value: wt


title: saline_dmso_2


title: DARLIN_v1_tissues_LL195-kidney

value: kidney


title: lps_inhibitors_1


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


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


title: DARLIN_v1_tissues_LL195-intestine

value: intestine


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


title: DARLIN_v1_tissues_LL195-liver

value: liver


title: B2M exon2 - LNPs 2.0 ug - Rep6

value: B2M exon2

value: LNP


title: saline_inhibitors_1

value: CD45+ cells

value: 5-6 months old


title: B2M exon2 - RNP 25 pmol - Rep3

value: RNP


title: B2M exon2 - LNPs 2.0 ug - Rep5


title: lps_dmso_1

value: brain

value: LPS

value: DMSO

value: Male


title: B2M exon2 - LNPs 2.0 ug - Rep4

value: Cutured HSPC


title: DARLIN_v1_tissues_LL193-spleen

value: spleen

value: CC


title: saline_dmso_1

value: Saline


title: B2M exon2 - LNPs 2.0 ug - Rep3


title: DARLIN_v1_tissues_LL193-ovary

value: ovary


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


title: EMT6 rep 1 [Mibio Medical]

city: Bothell

first: Alyson

middle: J

last: Smith

state: WA

address: 21717 30th Drive S.E., Building 3

department: Research

institute: Seagen Inc.

description: tpm.all.final.txt

data_processing:
FASTQ data files containing raw reads for each of the three samples were generated and analyzed separately by the vendor to generate single-cell feature (gene) counts using the function “cellranger count” from 10x Genomics Cell Ranger 5.0.0
A single feature count matrix aggregating across all three samples was generated using “cellranger aggr” from 10x Genomics Cell Ranger 6.0.0
From the resulting matrix, cells that had fewer than 1000 total counts; or more than 25% of their counts going to mitochondrial genes, or which had zero CD3 expression (zero counts for CD3D, CD3E, and CD3G), were excluded.
The reduced matrix was reanalyzed using “cellranger reanalyze”
Assembly: hg38
Supplementary files format and content: barcodes.tsv.gz is a compressed file containing the cell names for the scRNA-seq data.
Supplementary files format and content: features.tsv.gz is a compressed file containing the gene names for the scRNA-seq data.
Supplementary files format and content: matrix.mtx.gz is a compressed file containing the cell by gene count matrix for the scRNA-seq data.
FASTQ files from RNA-seq of all models were uniformly processed in-house using a  pipeline consisting of adapter trimming using Cutadapt v1.16;
read alignment was  performed using STAR v2.5.2b, to the mm10/GRCm38 genome and transcriptome  annotated by GENCODE and downloaded from UCSC
Isoform and gene-level transcript  quantification was conducted using RSEM v1.2.31
The FFPE samples for the in-house  CT26 model were run using RSEM parameters for a TruSeq stranded library (--forward- prob=0)
As a normalization step, we perfomed a rescaling of the gene- and isofrom-level quantification TPMS to exclude nonprotein-coding genes, a step that we have founf to improve the clustering of samples with mixed library prepartaion methods
Assembly: mm10
Supplementary files format and content: tpm.all.final.txt is text file containing per-sample gene-level quantification, in units of TPM (transcripts per million reads).  Columns are sample IDs, rows are genes, with a header row, and tab-separated fields.

source_name: Tumor

extract_protocol:
Frozen samples of primary human lung tumors underwent dead cell removal and T cell enrichment using the Sony MA900 Cell Sorter with a DAPI live/dead cell discriminator and staining for CD45 and CD3. Following dead cell removal and T cell enrichment (CD45+ 972 CD3+), cell count and viability were assessed using acridine orange/propidium iodide on a Nexcelom Cellometer and cells were washed twice in PBS + 0.04% bovine serum albumin. Following successful isolation, 15,000 cells per sample were loaded into the 10X Chromium chip using the Chromium Single Cell (v3.1 chemistry) with the intention of targeting 10,000 cells/sample for sequencing.
3′ GEX scRNA-Seq libraries were prepared according to manufacturer recommendations. Libraries were quality controlled on the Agilent TapeStation. Libraries were pooled and sequenced on the NovaSeq 6000 S2 100 flow cell targeting an average depth of 25,000 reads per cell (×10,000 cells/sample) or 250 million reads/sample.

value: Tumor

tag: source

value: Mibio Medical


title: E0771 rep 1 [Mibio Medical]

description: tpm.all.final.txt


title: DARLIN_v1_tissues_LL193-liver

value: liver


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

source_name: C57BL/6J brain


title: B2M exon2 - LNPs 2.0 ug - Rep2

value: B2M exon2


title: B2M exon2 - LNPs 2.0 ug - Rep1

value: LNP


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


title: DARLIN_v1_tissues_LL193-lung

value: lung


title: B2M exon2 - LNPs 1.5 ug - Rep6


title: Renca rep 3 [Champions Oncology]

description: tpm.all.final.txt

value: Renca

value: Champions Oncology


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

value: brain


title: DARLIN_v1_tissues_LL193-intestine

value: intestine


title: Renca rep 1 [Champions Oncology]

description: tpm.all.final.txt


title: DARLIN_v1_tissues_LL193-kidney

value: kidney


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


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


title: Renca rep 2 [Champions Oncology]

description: tpm.all.final.txt


title: DARLIN_v1_diversity_LL211-G

source_name: Granulocyte

value: Granulocyte


title: B2M exon2 - LNPs 1.5 ug - Rep4

value: Cutured HSPC


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


title: MC38 rep 3 [Champions Oncology]

description: tpm.all.final.txt


title: B2M exon2 - LNPs 1.5 ug - Rep5


title: B2M exon2 - LNPs 1.5 ug - Rep3


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


title: MC38 rep 2 [Champions Oncology]

description: tpm.all.final.txt


title: DARLIN_v1_diversity_LL207-G


title: B2M exon2 - LNPs 1.5 ug - Rep2


title: MC38 rep 1 [Champions Oncology]

description: tpm.all.final.txt


title: DARLIN_v1_diversity_LL206-G


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


title: LLC rep 6 [Champions Oncology]

description: tpm.all.final.txt

value: LLC


title: B2M exon2 - LNPs 1.5 ug - Rep1


title: DARLIN_v1_diversity_LL208-G

value: CC


title: DARLIN_v1_diversity_LL205-G


title: B2M exon2 - RNP 50 pmol - Rep6

value: RNP


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


title: LLC rep 5 [Champions Oncology]

description: tpm.all.final.txt


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


title: B2M exon2 - RNP 50 pmol - Rep4


title: DARLIN_v1_diversity_LL196-G


title: B2M exon2 - RNP 50 pmol - Rep5


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


title: DARLIN_v1_diversity_LL195-G


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


title: LLC rep 2 [Champions Oncology]

description: tpm.all.final.txt


title: B2M exon2 - RNP 50 pmol - Rep3


title: DARLIN_v1_diversity_LL193-G


title: LLC rep 1 [Champions Oncology]

description: tpm.all.final.txt


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


title: EMT6 rep 6 [Champions Oncology]

description: tpm.all.final.txt


title: B2M exon2 - LNPs 1.0 ug - Rep6


title: LLC rep 3 [Champions Oncology]

description: tpm.all.final.txt


title: DARLIN_v1_Gr_allele_bank_LL646-Gr-rep6


title: DARLIN_v1_diversity_LL200-G


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


title: B2M exon2 - RNP 50 pmol - Rep1


title: EMT6 rep 5 [Champions Oncology]

description: tpm.all.final.txt


title: B2M exon2 - LNPs 1.0 ug - Rep4


title: EMT6 rep 3 [Champions Oncology]

description: tpm.all.final.txt


title: B2M exon2 - LNPs 1.0 ug - Rep5


title: LLC rep 4 [Champions Oncology]

description: tpm.all.final.txt


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


title: B2M exon2 - RNP 50 pmol - Rep2


title: B2M exon2 - LNPs 1.0 ug - Rep3


title: DARLIN_v1_Gr_allele_bank_LL646-Gr-rep4


title: DARLIN_v1_Gr_allele_bank_LL646-Gr-rep8


title: DARLIN_v1_Gr_allele_bank_LL646-Gr-rep5


title: DARLIN_v1_Gr_allele_bank_LL646-Gr-rep3


title: M1-Right-19-Neuron-H3K27me3-R2


title: B2M exon2 - LNPs 1.0 ug - Rep2


title: DARLIN_v1_Gr_allele_bank_LL646-Gr-rep7


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


title: EMT6 rep 4 [Champions Oncology]

description: tpm.all.final.txt


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


title: DARLIN_v1_Gr_allele_bank_LL646-Gr-rep2


title: M1-Right-19-Glia-H3K27me3-R2


title: B2M exon2 - LNPs 1.0 ug - Rep1


title: M1-Right-19-Glia-H3K27me3-R1


title: DARLIN_v1_Gr_allele_bank_LL646-Gr-rep1


title: HSPCs_day14_smallRNA

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

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

data_processing:
For smallRNA-seq during erythroid differentiation: Raw reads with adapter sequence were trimmed by cutadapt with the parameters. Clean reads were then aligned to the Homo sapiens genome using Bowtie with default parameters. We used miRbase v21 annotation to count miRNA expression using featureCounts. Only reads with mapping quality score >30 were retained for further analysis by SAMtools.
genome build: Ensembl GRCh38.p12
processed data files format and content: mature miRNA raw counts text file

source_name: CD34+ cell

growth_protocol: For smallRNA-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 using Trizol reagent.
Small RNA libraries were generated using the NEBnext small RNA library preparation kit according to instructions (E7580S, New England BioLabs).

tag: lncrna probes

value: cord blood CD34+ derived erythroid cells

tag: sequencing type

value: smallRNA-seq


title: CT26 rep 3 [Champions Oncology]

description: tpm.all.final.txt

tag: tissue

value: Tumor


title: M1-Right-19-Neuron-H3K27me3-R1

tag: strain


title: M1-Right-18-Glia-H3K27me3-R2


title: DARLIN_SC_blood_LL837-spleen-CC

value: spleen


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

source_name: Peripheral Blood

value: Peripheral Blood

value: B2M exon2


title: K562_smallRNA

description: smallRNA-seq in K562 cell line

data_processing:
For smallRNA-seq in K562 cell line: Raw reads with adapter sequence were trimmed by cutadapt with the parameters. Clean reads were then aligned to the Homo sapiens genome using Bowtie with default parameters. We used miRbase v21 annotation to count miRNA expression using featureCounts. Only reads with mapping quality score >30 were retained for further analysis by SAMtools.
genome build: Ensembl GRCh38.p12
processed data files format and content: mature miRNA raw counts text file

source_name: K562 cell line

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

extract_protocol:
The cells were collected and RNA was harvested using Trizol reagent.
Small RNA libraries were generated using the NEBnext small RNA library preparation kit according to instructions (E7580S, New England BioLabs).

value: K562 cell line


title: EMT6 rep 2 [Champions Oncology]

description: tpm.all.final.txt


title: M1-Right-18-Glia-H3K27me3-R1


title: CT26 rep 2 [Champions Oncology]

description: tpm.all.final.txt


title: EMT6 rep 1 [Champions Oncology]

description: tpm.all.final.txt


title: DARLIN_SC_blood_LL837-skull-TC

source_name: skull

value: skull

value: TC


title: DARLIN_SC_blood_LL837-spleen-TC

source_name: spleen


title: M1-Right-18-Neuron-H3K27me3-R1

value: brain


title: CT26 rep 1  [Champions Oncology]

description: tpm.all.final.txt

value: Champions Oncology


title: M1-Right-18-Neuron-H3K27me3-R2


title: DARLIN_SC_blood_LL837-spleen-RC

value: RC


title: 4T1 rep 6 [Champions Oncology]

description: tpm.all.final.txt


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


title: HSPCs_day11_smallRNA

description: smallRNA-seq during erythroid differentiation


title: 4T1 rep 4 [Champions Oncology]

description: tpm.all.final.txt


title: M1-Right-17-Neuron-H3K27me3-R1


title: M1-Right-17-Neuron-H3K27me3-R2


title: 4T1 rep 3 [Champions Oncology]

description: tpm.all.final.txt


title: HSPCs_day18_smallRNA


title: 4T1 rep 5 [Champions Oncology]

description: tpm.all.final.txt


title: DARLIN_SC_blood_LL837-skull-RC


title: 4T1 rep 2 [Champions Oncology]

description: tpm.all.final.txt


title: M1-Right-17-Glia-H3K27me3-R2


title: U87.mRNA

city: Jerusalem

first: Yifat

last: Edrei

address: Ein Karem Cmpus

country: Israel

institute: The Hebrew University of Jerusalem

data_processing:
Library strategy: STARR-seq
DNA and cDNA samples were sequenced using the Illumina HiSeq2500 device, 125bp paired-end protocol. Technical and biological replications performed using illumina MiSeq, 250bp paired-end
Alignment with the hg19 reference genome was performed on the first 40 bp from both sides of the DNA segments, using Bowtie2
Reads with mapping quality value above 40 aligned with the probe targets were considered for further analyses. Each of the captured genomic segments was given a unique ID according to genomic location and indicated the total number of DNA and RNA reads
Genome_build: hg19
Supplementary_files_format_and_content: text

molecule: polyA RNA

source_name: post-methylation cDNA

growth_protocol: Human GBM T98G/U87 cells were cultured in minimum essential medium-Eagle (Biological Industries), supplemented with 10% heat-inactivated FBS #04-127-1A (Biological Industries), 1% penicillin/streptomycin P/S # 03-031-1B (Biological Industries), 1% L-glutamine #03-020-1C (Biological Industries;), 1% nonessential amino acids, #01-340-1B (Biological Industries) and 1% sodium pyruvate #03-042-1B (Biological Industries), at 37°C and 5% CO2.

extract_protocol:
Plasmid DNA was extracted from 2.5x10e7 cells, 24 h post-transfection. Cells were rinsed twice with PBS pH 7.4, using the NucleoSpin Plasmid EasyPure kit #740727250 (Macherey-Nagel), according to the manufacturer’s protocol. Total RNA was extracted from 2.5x10e7 cells 24h post-transfection using GENEZOL reagent # GZR200 (Geneaid), according to the manufacturer’s protocol. The polyA+ RNA fraction was isolated using Dynabeads Oligo-(dT)25 #61002 (Thermo scientific), scaling up the manufacturer’s protocol 5-fold per tube, and treated with 10 U turboDNase #AM2238 (Invitrogen) at 20 ng/µl 37°C, for 1 h. Two reactions of 50 µl each, were  pooled  and subjected  to  RNeasy  MinElute clean  up kit  #74204 reaction  (Qiagen)  to inactivate turbo DNase and concentrate the  polyA+ RNA.
First  strand  cDNA  synthesis  was  performed  with  1-1.5 µg polyA+ RNA  in a total  of  four  reactions, 20 µl each, using the  Verso cDNA Synthesis Kit #AB1453B (Thermo scientific ) according to the manufacturer’s protocol, with  a  reporter-RNA  specific primer (5’-CAAACTCATCAATGTATCTTATCATG-3’). cDNA (50 ng) was first amplified by PCR, at 98°C for 3 min, followed by 15 cycles at 95°C for 20s each, 65°C for 15s, 72°C for 30s. Final extension was performed at 72°C for 2 min, using the Hifi Hot Start Ready Mix (KAPA), with reporter-specific primers. Forward primer: 5’-GGCCAGCTGTTGGGGTG*T*C*C*A*C-3’ which spans the splice junction of the synthetic intron, and reverse primer: 5’-CTTATCATGTCTGCTCGA*A*G*C-3’, where “*” indicates phosphorothioate bonds. In total, 16-20 reactions were performed. The amplified products were purified with 0.8 x Agencourt AMPureXP DNA beads and eluted in 20 µl nuclease- free water. The resultant purified products served as a template for a second PCR performed under the following conditions: 95°C for 3 min, 12 cycles of 98°C for 15s, 65°C for 30s, 72°C for 30s. Final extension was performed at 72°C for 2 min, with forward Ilumina universal primer: 5’-TAGAGCATGCACCGGTAATGATACGGCGACCACCGAGATCT-3’ and reverse Indexed Ilumina primer: 5’-GGCCGAATTCGTCGACCAAGCAGAAGACGGCATACGAGAT-3’. PCR products were purified with 0.8 x Agencourt AMPureXP DNA beads, eluted in 10 μl nuclease-free water, and pooled.

treatment_protocol: The pGL3-promoter vector (Promega, GenBank accession number U47298) was modified by replacing the sequence between SacI and AfeI with a synthetic sequence 33 and then insertion of the capture library fragments (between AgeI-HF and SalI-HF). Sample #AK100 capture library was selected for this construction. 20 µg of the plasmid DNA were transfected into 2x10e6 cells at 70-80% confluence, using the Lipofectamine 3000 transfection kit #L3000-015 (Invitrogen), according to the manufacturer’s protocol. In each experiment, 5x10e7 cells were transfected and incubated at 37°C, for 24 h.

value: Bisulfite

tag: sequenced molecule

value: post-methylation cDNA


title: U87.mDNA

source_name: post-methylation genomic DNA

value: post-methylation genomic DNA


title: DARLIN_SC_blood_LL837-skull-CC

tag: cell type

value: CC


title: U87.RNA

source_name: cDNA

value: cDNA


title: DARLIN_SC_blood_LL837-lung-CC

value: lung


title: DARLIN_SC_blood_LL837-lung-TC


title: U87.DNA

source_name: genomic DNA

value: genomic DNA


title: CT26 rep 6 [Seagen]

description: tpm.all.final.txt

value: Tumor

value: Seagen


title: 4T1 rep 1 [Champions Oncology]

description: tpm.all.final.txt


title: M1-Right-16-Neuron-H3K27me3-R1


title: DARLIN_SC_blood_LL837-lung-RC


title: T98g.mRNA.techRep


title: T98g.mDNA.techRep


title: M1-Right-16-Neuron-H3K27me3-R2


title: T98g.RNA.techRep


title: DARLIN_SC_blood_LL837-liver-TC

source_name: liver

value: liver


title: CT26 rep 2 [Seagen]

description: tpm.all.final.txt


title: CT26 rep 4 [Seagen]

description: tpm.all.final.txt


title: T98g.DNA.techRep


title: M1-Right-16-Glia-H3K27me3-R2


title: DARLIN_SC_blood_LL837-liver-RC


title: DARLIN_SC_blood_LL837-LF-TC

source_name: LeftLeg

value: LeftLeg


title: IgD- B, 34941, week 6, plate 7

city: Bethesda

first: Chaim

middle: A

last: Schramm

state: MD

address: 40 Convent Dr

department: Vaccine Research Center

institute: NIAID, NIH

description: poly A RNA via SmartSeq

data_processing:
The counts matrix was generated with Cellranger v3.1.0, using the protein-coding genes of the rhesus macaque reference genome from Ensembl (Mmul10, release #101). The filtered feature/barcode matrices were imported into Seurat v4.0.1 using R v4.0.4. Cells absent from either the gene expression or antibody capture datasets were eliminated, along with cells expressing zero counts of hash oligos. For hash demultiplexing, Seurat’s HTOdemux function was applied, and only singlets were retained for subsequent analyses. The filters based on percent mitochondrial genes and number of genes expressed varied between sample, but the upper threshold for percent mitochondrial genes ranged from 4-10%; the minimum for number of genes expressed was 200 genes for each sample; the upper threshold for number of genes expressed ranged from 1500-2500. The raw counts of each individual sample were merged.  For the innate cell sorts, clusters of contaminating B and T cells (identified using Seurat’s FindClusters and RunUMAP) were removed and not included in downstream analysis. The merged and filtered data matrix was then corrected for sparseness using DeepImpute with tensorflow v2.1.0 and default parameters. The imputed matrix was then re-imported into Seurat.  For both innate and T cells, Seurat was used to normalize the (imputed) count matrix, find variable genes, and regress out technical factors. Principle compents (PCs) were then calculated and used as input to Harmony to remove batch effects between samples. The integrated output was clustered using FindClusters and marker genes were identified with FindAllMarkers. Canonical marker genes were used to annotate the detected clusters.  Gene set enrichment analysis of the innate cell data was conducted using the R library fgsea. The data were broken down by cell type and time point, and differential expression analysis of all 21,369 genes was conducted using FindMarkers between each subset. Finally, the statistically significant (adjusted p < 0.05) pathways were detected by inputting the genes, ranked by average log fold change, into the function fgsea. The Hallmark gene set databases originated from MSigDB.  Signature scores for 50 pro-inflammatory genes previously determined to be predictive of antibody responses to a variety of vaccines were calculated using AddModuleScore in Seurat.
cellranger vdj (10x Genomics) was used to assign TCR annotations for each read-pair. TCR alpha recovery was minimal and not followed up further. An in-house shell script was used to filter for productive TCR betas. All subsequent analyses were done in R (v4.0.2). Cells with duplicated barcodes were discarded, and the TCR data was merged with the T cell transcriptomics Seurat (v4.0.1) object metadata for hash demultiplexing. Usage of each TRBV gene was compared between the S-specific and non-specific T cells from each animal using a paired samples Wilcoxon test in R.
SmartSeq sequencing reads were trimmed with Trimmomatic v 0.39, trimming nucleotides with Phred score below 20 and removing reads with resulting length less than 50 nucleotides. Alignment of reads to macaque protein-coding reference build Mmul10 was performed using Spliced Transcript to a Reference (STAR) v. 2.5.1b. Gene quantifications of reads as transcripts per million (TPM) were computed with Stringtie v. 1.33. Individual cells were removed from the analysis based on trimming and alignment metrics, as well as read quantity, feature quantity, quantity of mitochondrial reads, and diversity of transcripts.   Seurat was used to calculate principal component analysis, which was used as input to integrate the data by batch with Harmony. PCA, cell clustering and UMAP dimensionality reductions were conducted in Seurat. A single cluster was identified as likely T cells by its more than triple the expression of CD3E than the next highest cluster and so was removed from the analysis. Clustering and dimensionality reduction was repeated after this cell filter.   Differential expression of cell clusters was conducted with Seurat's function FindAllMarkers, and FindMarkers was used for comparison of timepoints week 2 and week 6. Those results were used as input to Gene set enrichment analysis conducted with the R package fgsea and the hallmark pathwaysfrom MSigDB.
Full-length V(D)J sequences from were reconstructed from SmartSeq data using BALDR and filtered using filterBALDR.pl (https://github.com/scharch/filterBALDR). Gene annotation and clonal assignment were done using SONAR v4.2 in single cell mode. Clonal flow diagrams were plotted using the ggalluvial package in R v4.2.1. Public clone were identified by clustering CDR H3 amino acid sequences using usearch as previously described . Members of the human public clone were identified from CovAbDab sequences downloaded as of January 24th, 2023 meeting the following criteria: (1) derived from IGHV3-30, IGHV3-30-3, or IGHV3-30-5; (2) 14 amino acid CDR H3 with Gly at position 6 and Tyr at position 8; (3) originating from a human; (4) not annotated as binding to any RBD or NTD epitope. This resulted in 326 public clone sequences, which were plotted using ggseqlogo.
Assembly: Mmul10
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz
Supplementary files format and content: tab-delimited HTO assignments for each cell
Supplementary files format and content: comma-delimited values of gene expression in TPM
Supplementary files format and content: AIRR-formated VDJ sequences recovered from B cells
Supplementary files format and content: 10x Genomics output files: TR VDJ contigs with annotations
Supplementary files format and content: RDS-formatted R data files with complete Seurat objects

organism: Macaca mulatta

source_name: PBMC

extract_protocol:
Frozen rhesus macaque PBMCs were thawed into warm R10 media (RPMI + 10% Fetal Bovine Serum + 2 mL L-Glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin; all reagents from Gibco) containing DNase I (MilliporeSigma), followed by one wash with R10 and one wash with FACS buffer (PBS with 2% FBS). For B and innate cell staining, cells were resuspended in 100µL of Live/Dead Fixable Blue Dead Cell Stain Kit (Invitrogen, cat# L23105) diluted 1:200 in PBS for 10 min at room temperature. Cells were washed with FACS buffer and incubated for 20 min with the staining cocktail consisting of antibodies and probes. Additionally, each sample was labelled with 1µl of TotalSeq-C Hashtag antibodies (Biolegend) that was incubated together with the staining cocktail. After incubation, cells were washed twice with FACS buffer and resuspended in R10 for sort.  From each sample, 10,000-60,000 innate cells were sorted into a tube containing FBS according to the gating strategy showed in Fig. S1B. From baseline samples, 50 thousand naïve B cells were also sorted in a separate tube. From week 2 and 6 samples, antigen-specific cells were single-cell sorted into 96-well plates containing 5 µL of TCL buffer (Qiagen) with 1% b-mercaptoethanol for sequencing by SmartSeq. All sorts were performed using a BD FACSymphony S6 Cell Sorter (BD Biosciences) with BD FACSDiva Software version 9.5.1 (BD Biosciences) and data were analyzed using Flowjo v10.8.1.
Frozen rhesus macaque PBMC were thawed into warm R10 media (RPMI + 10% Fetal Bovine Serum + 2 mL L-Glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin; all reagents from Gibco) containing DNase I (MilliporeSigma) and rested for 1 hour at 37oC / 5% CO2.  Cells were stimulated with two peptide pools corresponding to S1 and S2 of the vaccine insert SARS-CoV-2 S protein (JPT Peptide Technologies) at 2 µg/mL of each peptide for 6 hours at 37oC / 5% CO2.  A DMSO only control was included for each sample.  Anti-CD154 BV421(Biolegend, clone 24-31, cat# 310824) was included during the 6-hour culture and GolgiStop (BD Biosciences) was added after 2 hours of stimulation.  Following stimulation, cells were washed and stained with Aqua LIVE/DEAD dye (ThermoFisher) for 10 minutes, and subsequently stained with antibody mix. In addition, cells were incubated with TotalSeqTM Hashtag antibodies (Biolegend) with separate antibodies applied to the DMSO only condition and the stimulated conditions. For each animal, 1000-2000 memory CD4 cells from the DMSO only condition were sorted and combined with memory cells expressing CD154 and CD69 from each S pool stimulation for processing for 10X Genomics.
Bulk-sorted cells were pooled into a tube and loaded on the 10x Genomics Chromium Chip according to the manufacturer’s protocol for the Next GEM Single Cell 5’ Kit v1.1 (10x Genomics, PN-1000165). To sequence single-cell gene expression and cell surface oligonucleotides (from Hashtag antibodies), libraries were prepared according to manufacturer’s instructions using the Chromium Single Cell 5' Library Construction Kit (10x Genomics, PN-1000020) and Chromium Single Cell 5' Feature Barcode Library Kit (10x Genomics, PN-1000080), respectively. To sequence TCR repertoire libraries were prepared using the Chromium Single Cell V(D)J Enrichment Kit (10x Genomics, PN- PN-1000005). These libraries were sequenced on an Illumina NovaSeq platform (Illumina). To sequence BCR repertoire, heavy and light chains were amplified from the cDNA using IgG_REV, IgA_REV, IgK_REV or IgL_REV primers (Table S3) with the addition of Illumina sequences as detailed in (25). These Illumina-ready libraries were sequenced using 2x300 paired-end reads on an Illumina MiSeq
Full-length transcriptomes were sequenced using a modified SMART-Seq protocol. Heavy and light chain variable regions were enriched by amplifying cDNA with a forward primer complementary to the TSO and IgA/IgG_REV or IgK/IgL_REV primer pools.
OTHER: scRNA-seq with FeatureSeq and scAIRR-seq

value: PBMC

value: IgD- memory B cells

tag: library type

value: mRNA

tag: tags

value: Nextera UDI set A

value: mRNA-1273 vaccination

tag: time

value: week 6


title: CT26 rep 3 [Seagen]

description: tpm.all.final.txt

tag: cell line


title: M1-Right-16-Glia-H3K27me3-R1

value: brain


title: IgD- B, 34941, week 6, plate 8

description: poly A RNA via SmartSeq

value: Nextera UDI set D

tag: treatment


title: CT26 rep 1 [Seagen]

description: tpm.all.final.txt


title: M1-Right-15-Glia-H3K27me3-R2


title: DARLIN_SC_blood_LL837-LF-CC

tag: carlin protocol

tag: carlin locus


title: M1-Right-15-Neuron-H3K27me3-R2


title: M1-Right-17-Glia-H3K27me3-R1


title: CT26 rep 5 [Seagen]

description: tpm.all.final.txt


title: Renca rep 6 [Seagen]

description: tpm.all.final.txt

value: Renca


title: M1-Right-15-Neuron-H3K27me3-R1


title: IgD- B, 16C237, week 6, plate 2

description: poly A RNA via SmartSeq

value: Nextera UDI set B


title: DARLIN_SC_blood_LL837-LF-RC

value: RC


title: IgD- B, 36186, week 6, plate 4

description: poly A RNA via SmartSeq


title: DARLIN_SC_blood_LL837-liver-CC

value: CC


title: IgD- B, 16C303, week 6, plate 7

description: poly A RNA via SmartSeq


title: Renca rep 5 [Seagen]

description: tpm.all.final.txt


title: Cas9/CARLIN_diversity_SB838-G

value: Granulocyte


title: Cas9/CARLIN_diversity_SB822-G


title: Renca rep 4 [Seagen]

description: tpm.all.final.txt

value: Tumor


title: Cas9/CARLIN_diversity_SB842-G


title: M1-Right-14-Neuron-H3K27me3-R1


title: IgD- B, 16C301, week 6, plate 3

description: poly A RNA via SmartSeq


title: Renca rep 3 [Seagen]

description: tpm.all.final.txt

value: Seagen


title: Renca rep 1 [Seagen]

description: tpm.all.final.txt


title: IgD- B, 16C303, week 6, plate 8

description: poly A RNA via SmartSeq

value: Nextera UDI set C


title: M1-Right-14-Neuron-H3K27me3-R2


title: IgD- B, 16C237, week 6, plate 8

description: poly A RNA via SmartSeq


title: M1-Right-15-Glia-H3K27me3-R1


title: IgD- B, 16C283, week 6, plate 2

description: poly A RNA via SmartSeq


title: M1-Right-14-Glia-H3K27me3-R1


title: Cas9/CARLIN_diversity_SB837-G


title: M1-Right-13-Glia-H3K27me3-R2


title: A20 rep 1 [Seagen]

description: tpm.all.final.txt


title: IgD- B, 16C222, week 6, plate 3

description: poly A RNA via SmartSeq


title: IgD- B, 16C283, week 6, plate 1

description: poly A RNA via SmartSeq


title: MC38 rep 1 [Seagen]

description: tpm.all.final.txt


title: M1-Right-14-Glia-H3K27me3-R2


title: IgD- B, 16C283, week 6, plate 3

description: poly A RNA via SmartSeq


title: A20 rep 2 [Seagen]

description: tpm.all.final.txt


title: Single oocyte, MII5

first: Yi-Rong

last: Jiang

state: Zhejiang

address: Room C306, Chemistry Building, Zijingang Campus, No. 866 Yuhangtang Ro

department: Department of chemistry

institute: Zhejiang University

description: MATQ-seq

data_processing:
The raw transcriptome sequencing data were trimmed using Cutadapt (version 4.0) to remove primer sequences, followed by trimming of the extra bases generated by dC-tailing.
The reads were then mapped to the genome using STAR software (version 2.7.10a).
Gene annotations were performed using Gene Code Annotation Release M29 (GRCm39, GENCODE).
After retrieving the mapping information of the reads, feature counts were used to count the gene expression level.
The numbers of total reads were summarized to normalize the gene expression. The unit of RPM is defined as the reads per one million total reads. Gene expression level is defined by the number of reads of a gene divided by the total number of reads of all genes and multiplied by 1,000,000.
Assembly: m39
Supplementary files format and content: tsv files includes raw counts for each samples.

source_name: Oocyte

extract_protocol:
To collect the oocytes at fully grown GV stage, around six-weeks-old to eight-weeks-old female mice were injected with 7 IU of pregnant mare serum gonadotropin (PMSG) and humanely sacrificed 44 h later. Then the oocytes at fully grown GV stage were collected in the M2 medium. For collecting the oocytes arrested in second meiotic metaphase (MII stage), approximately 48 h after PMSG (7 IU) injection, 7 IU of human chorionic gonadotropin was injected into female mice. After an additional 20 h, cumulus-oocyte complexes (COCs) were surgically removed from fallopian tubes. Then the oocytes at MII stage were collected in the M2 medium from COCs after digestion of 300 IU/mL hyaluronidase.
In the reverse transcription, 2.4 μL of the reverse transcription mix containing 0.8 μL of 5× First Strand Buffer, 0.2 μL of 0.1 M DTT, 0.1 μL of RNaseOUT, 0.15 μL of Superscript III and 1.15 μL of RNase-free water was added into each droplet, and the droplets were treated with ten cycles of annealing (ramping from 8 °C to 50 °C) on the thermocycler. After reverse transcription, the residual primers were digested by T4 polymerase at 37 °C for 40 min and 80 °C for 20 min, and then the RNAs were digested by RNase-H and RNase-If at 37 °C for 15 min and 72 °C for 15 min. Next, dC-tailing and second strand synthesis were performed according to the protocol described in the MATQ-seq technique. The PCR amplification of the droplets were conducted with a 28-cycle PCR program (95 °C for 15 s, 60 °C for 20 s, and 72 °C for 2 min). After PCR amplification, pooled libraries were purified with 1.2X AMPure XP beads into PCR-grade water. The ABclonal Rapid Plus DNA Lib Prep kit of Illumina was used to construct the PCR product library according to the protocol provided with the kit. The cDNA was firstly sheared to 300 bp using a Covaris S220 sonicator. End repair and A-tailed was then performed using a NGS Fast DNA Library Prep kit of Illumina. T4 DNA ligase was used to ligate adaptors to samples. Libraries were then diluted and sequenced on a NovaSeq 6000 machine by Haprolos, using the NovaSeq S4 reagent kit v1.5 (300 cycles).

value: Oocyte

tag: developmental stage

value: MII


title: Cas9/CARLIN_SC_readout_SB_Cas9_OC_Tr

source_name: Tr

value: Tr


title: IgD- B, 36186, week 6, plate 1

description: poly A RNA via SmartSeq

value: PBMC

value: IgD- memory B cells

value: mRNA


title: Cas9/CARLIN_SC_readout_SB_Cas9_OC_Ta

source_name: Ta

value: Ta


title: Cas9/CARLIN_diversity_SB821-G


title: M1-Right-13-Glia-H3K27me3-R1

source_name: C57BL/6J brain

value: brain


title: Camellia-seq-lineage_LL744-P2-RC

source_name: AGM-HSC

value: AGM-HSC


title: M1-Right-12-Neuron-H3K27me3-R1


title: Single oocyte, MII6

description: MATQ-seq


title: M1-Right-13-Neuron-H3K27me3-R2


title: Cas9/CARLIN_diversity_SB820-G

source_name: Granulocyte


title: Camellia-seq-lineage_LL744-P2-CC


title: M1-Right-12-Glia-H3K27me3-R2


title: IgD- B, 36186, week 6, plate 3

description: poly A RNA via SmartSeq

value: mRNA-1273 vaccination

value: week 6


title: Cas9/CARLIN_SC_readout_SB_Cas9_OC_H

source_name: H

value: H


title: Single oocyte, MII2

description: MATQ-seq


title: M1-Right-13-Neuron-H3K27me3-R1


title: IgD- B, 16C301, week 6, plate 1

description: poly A RNA via SmartSeq

value: Nextera UDI set B


title: IgD- B, 16C301, week 6, plate 2

description: poly A RNA via SmartSeq


title: Single oocyte, MII3

description: MATQ-seq


title: IgD- B, 16C235, week 6, plate 6

description: poly A RNA via SmartSeq

value: Nextera UDI set A


title: Camellia-seq-lineage_LL744-P1-TC

value: TC


title: M1-Right-12-Neuron-H3K27me3-R2


title: M1-Right-11-Neuron-H3K27me3-R2


title: M1-Right-12-Glia-H3K27me3-R1


title: Camellia-seq-lineage_LL744-P1-RC

value: RC


title: Single oocyte, MII4

description: MATQ-seq


title: Single oocyte, GV9

description: MATQ-seq

value: GV


title: IgD- B, 16C235, week 6, plate 4

description: poly A RNA via SmartSeq

value: Nextera UDI set C


title: Single oocyte, MII1

description: MATQ-seq

value: Oocyte

value: MII


title: Camellia-seq-lineage_LL744-P1-CC

value: CC


title: Camellia-seq-lineage_LL731-P3-TC_rep2

source_name: BoneMarrow-HSC

value: BoneMarrow-HSC


title: M1-Right-11-Neuron-H3K27me3-R1


title: Single oocyte, GV8

description: MATQ-seq


title: IgD- B, 16C222, week 2, plate 3 & 16C283, week 2, plate 7

description: poly A RNA via SmartSeq

value: PBMC

value: IgD- memory B cells

value: mRNA

value: 16C222_wk2_3/Nextera UDI set B ;

value: week 2


title: M1-Right-11-Glia-H3K27me3-R2

value: brain


title: Camellia-seq-lineage_LL731-P3-TC


title: Single oocyte, GV7

description: MATQ-seq


title: M1-Right-11-Glia-H3K27me3-R1


title: Single oocyte, GV5

description: MATQ-seq


title: IgD- B, 16C222, week 6, plate 1

description: poly A RNA via SmartSeq

value: Nextera UDI set D

value: mRNA-1273 vaccination

value: week 6


title: Camellia-seq-lineage_LL731-P3-RC_rep2


title: Single oocyte, GV6

description: MATQ-seq


title: IgD- B, 16C222, week 6, plate 2

description: poly A RNA via SmartSeq

value: Nextera UDI set A


title: Camellia-seq-lineage_LL731-P3-RC

value: RC


title: M1-Right-10-Neuron-H3K27me3-R1


title: M1-Right-10-Glia-H3K27me3-R2


title: Single oocyte, GV4

description: MATQ-seq

value: GV


title: M1-Right-10-Neuron-H3K27me3-R2


title: IgD- B, 36186, week 2, plate 2

description: poly A RNA via SmartSeq

value: Nextera UDI set C


title: Camellia-seq-lineage_LL731-P3-CC


title: IgD- B, 16C235, week 2, plate 1

description: poly A RNA via SmartSeq

value: Nextera UDI set B


title: IgD- B, 36186, week 2, plate 1

description: poly A RNA via SmartSeq


title: IgD- B, 16C235, week 6, plate 5

description: poly A RNA via SmartSeq


title: M1-Right-10-Glia-H3K27me3-R1


title: Single oocyte, GV1

description: MATQ-seq

value: Oocyte


title: IgD- B, 16C303, week 2, plate 2

description: poly A RNA via SmartSeq


title: Single oocyte, GV3

description: MATQ-seq


title: M1-Right-09-Neuron-H3K27me3-R2


title: M1-Right-09-Neuron-H3K27me3-R1


title: Camellia-seq-lineage_LL731-P2-TC_rep2

value: TC


title: Single oocyte, GV2

description: MATQ-seq


title: Camellia-seq-lineage_LL731-P2-TC

value: BoneMarrow-HSC


title: IgD- B, 16C303, week 2, plate 1

description: poly A RNA via SmartSeq

value: PBMC


title: M1-Right-09-Glia-H3K27me3-R2


title: Camellia-seq-lineage_LL731-P2-RC


title: Camellia-seq-lineage_LL731-P3-CC_rep2

value: CC


title: IgD- B, 34941, week 2, plate 2

description: poly A RNA via SmartSeq

value: IgD- memory B cells

value: mRNA


title: OT11-PBS-GEX_ASC1908A8

city: Pittsburgh

first: Tracy

last: Tabib

state: PA

address: 200 Lothrop Street

department: Department of Medicine

institute: University of Pittsburgh

description: 10x Genomics

data_processing:
Gene Expression (GEX) FASTQ files were aligned and a cell barcode x gene count matrix was generated with CellRanger 6, in reference to 10X Genomics' mouse refdata-gex-mm10-2020-A transcriptome.  BCR and TCR FASTQ files were aligned using CellRanger 6 to 10X Genomics' mouse V(D)J reference, vdj_GRCm38_alts_ensembl-5.0.0
Secondary analysis was performed with raw_feature_bc_matrix.h5 files for gene expression data and respective all_contig_annotations.csv and clonotypes.csv for TCR and BCR data. R 4.2.1 was utilized with the Seurat package (v4.3.0) and nichenetr (v1.1.0)
The raw_feature_bc_matrix.h5 files were initially loaded into Seurat and filtered such that only cells expressing at least 200 genes were retained.  Only cells containing less than 1% hemoglobin content were retained.
Wilcoxon rank sum test with Bonferroni FDR correction was used for DEG testing.
Assembly: refdata-gex-mm10-2020-A for transcriptome and vdj_GRCm38_alts_ensembl-5.0.0 for V(D)J reference
Supplementary files format and content: 10x Genomics output scRNA files: raw_feature_bc_matrix.h5
Supplementary files format and content: VDJ output files:  all_contig_annotations.csv and clonotypes.csv

source_name: Hindlimb muscle

extract_protocol: Llibraries were constructed according to the manufacturer's directions as described in 10X Genomics' Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index), Protocol CG000331

treatment_protocol: As outlined in the experimental protocol, scRNAseq was performed on muscle-resident/infiltrating cells isolated from mice immunized with vehicle (PBS) versus recombinant HRS protein.  No additional treatment was administered to experimental mice.

value: Hindlimb muscle

value: PBS

tag: chemistry


title: OT11-MA-TCR_ASC1908A2

description: 10x Genomics

value: MA


title: IgD- B, 34941, week 2, plate 3

description: poly A RNA via SmartSeq

value: Nextera UDI set D

value: mRNA-1273 vaccination

value: week 2


title: M1-Right-08-Neuron-H3K27me3-R2

value: brain


title: M1-Right-09-Glia-H3K27me3-R1


title: OT11-MA-GEX_ASC1908A6

description: 10x Genomics


title: Camellia-seq-lineage_LL731-P2-CC


title: OT11-MA-BCR_ASC1908A4

description: 10x Genomics


title: Camellia-seq-lineage_LL731-P2-CC_rep2


title: M1-Right-08-Neuron-H3K27me3-R1


title: IgD- B, 34941, week 2, plate 1

description: poly A RNA via SmartSeq


title: MYD88-PBS-GEX_ASC1908A11

description: 10x Genomics


title: IgD- B, 16C301, week 2, plate 1

description: poly A RNA via SmartSeq


title: M1-Right-08-Glia-H3K27me3-R2


title: Camellia-seq-lineage_LL731-P1-TC_rep2


title: IgD- B, 16C237, week 2, plate 1

description: poly A RNA via SmartSeq

value: Nextera UDI set A


title: MYD88-MA-GEX_ASC1908A9

description: 10x Genomics


title: Camellia-seq-lineage_LL731-P1-TC


title: MYD88-MA-TCR_ASC1908A13

description: 10x Genomics


title: M1-Right-08-Glia-H3K27me3-R1


title: Camellia-seq-lineage_LL731-P2-RC_rep2

value: RC


title: M1-Right-07-Neuron-H3K27me3-R2


title: Camellia-seq-lineage_LL731-P1-RC_rep2


title: Camellia-seq-lineage_LL731-P1-RC


title: CD4creMYD88-PBS-GEX_ASC1908A12

description: 10x Genomics

value: Cross of B6.Cg-Tg(Cd4-cre)1Cwi/BfluJ and B6.129P2(SJL)-Myd88tm1Defr/J


title: MYD88-MA-BCR_ASC1908A15

description: 10x Genomics


title: HeLa cells,NAA, day3, 2

city: Tianjin

first: Dongpeng

last: Zheng

state: Tianjin

address: No. 92, Weijin Road, Xuefu Street, Nankai District

department: School of Pharmaceutical Science and Technology

institute: Tianjin university

data_processing:
To remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (V1.9.1, phred cutoff: 20, error rate: 0.1, adapter overlap: 1bp, min. length: 75, proportion of N: 0.1) to be high quality clean data
reference genome sequences and gene model annotation files of relative species were downloaded from genome website, such as UCSC, NCBI, ENSEMBL. Secondly, Hisat2 (v2.0.1) was used to index reference genome sequence. Finally, clean data were aligned to reference genome via software Hisat2 (v2.0.1).
In the beginning transcripts in fasta format are converted from known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data.
Assembly: hg18
Supplementary files format and content: txt，HeLa_all.fpkm.txt

source_name: HeLa

growth_protocol: The human cell lines HeLa was cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin mixture PS at 37℃ in a 5% CO2 humidified atmosphere.

extract_protocol:
the total RNA was isolated from cells by using  total RNA purification kit (Tiangen, Cat# DP419)
1 μg total RNA was used for following library preparation. The poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexs were multiplexed and loaded on an Illumina HiSeq/ Illumina Novaseq/ MGI2000 instrument for sequencing using a 2x150 paired-end (PE) configuration according to manufacturer’s instructions.

treatment_protocol: Incubate all cells in 5% CO2 at 37 °C, and when cells have grown to a certain density, add 300 μM NAA and DMSO for 3 d, and then extract the total RNA.

value: HeLa

value: Human cervical cancer cells

value: 300 microM NAA treatment for 3 days


title: M1-Right-07-Neuron-H3K27me3-R1


title: HeLa cells,NAA, day3, 3


title: HeLa cells,NAA, day3, 1


title: M1-Right-07-Glia-H3K27me3-R2


title: M1-Right-07-Glia-H3K27me3-R1


title: Camellia-seq-lineage_LL731-P1-CC_rep2

value: BoneMarrow-HSC


title: CD4creMYD88-MA-TCR_ASC1908A14

description: 10x Genomics


title: Camellia-seq-lineage_LL731-P1-CC

value: CC


title: Camellia-seq-lineage_LL653-E6-P2-TC

source_name: FetalLiver-HSC

value: FetalLiver-HSC

value: TC


title: HeLa cells,DMSO, day3, 1

value: DMSO treatment for 3 days


title: HeLa cells,DMSO, day3, 2


title: HeLa cells,DMSO, day3, 3


title: CD4creMYD88-MA-GEX_ASC1908A10

description: 10x Genomics

value: Hindlimb muscle


title: Camellia-seq-lineage_LL653-E6-P2-RC


title: CD4creMYD88-MA-BCR_ASC1908A16

description: 10x Genomics

value: MA


title: C57-PBS-GEX_ASC1908A7

description: 10x Genomics

value: PBS


title: M1-Right-06-Glia-H3K27me3-R2


title: H9_rep2 [H9_2]

city: BEIJING

first: QINGYUAN

last: LIU

state: BEIJING

address: QINGHUAYUAN street，HAIDIAN distribute

institute: Tsinghua University

data_processing:
For RNA-seq data, sequenced reads were trimmed for adaptor sequence and low-quality sequence using Trim galore (v0.6.4).
Reads were mapped to human genome 38 using hisat2 (v2.2.0).
Gene count and TPM value were calculated by Stringtie (v2.1.4).
Assembly: hg38
Supplementary files format and content: The tab-delimited text files include raw counts for each sample.

extract_protocol:
Total RNA was extracted using the TRIzol® Reagent (Life Technologies).
For RNA-seq of WT DAZL and c.808C>T DAZL hPGCLCs at two time points, libraries were constructed by Genewiz. For RNA-seq of H9, libraries were constructed using VAHTS mRNA-seq v2 Library Prep Kit (Vazyme NR611-02).

value: hESCs

tag: genotype

value: WT H9

value: without treatment


title: C57-MA-TCR_ASC1908A1

description: 10x Genomics


title: Camellia-seq-lineage_LL653-E6-P1-TC

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

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).


title: M1-Right-06-Neuron-H3K27me3-R2

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

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

value: brain


title: H9_rep1 [H9_1]


title: C57-MA-GEX_ASC1908A5

description: 10x Genomics


title: Human Lung DTC rep 2 [Seagen]

description:
barcodes.tsv.gz
features.tsv.gz
matrix.mtx.gz

source_name: Tumor

value: Tumor

tag: source

value: Seagen


title: Camellia-seq-lineage_LL653-E6-P2-CC


title: Camellia-seq-lineage_LL653-E6-P1-RC

value: RC


title: M1-Right-05-Neuron-H3K27me3-R2


title: C57-MA-BCR_ASC1908A3

description: 10x Genomics


title: Renca rep 2 [Seagen]

description: tpm.all.final.txt

value: Renca


title: M1-Right-05-Neuron-H3K27me3-R1


title: Camellia-seq-lineage_LL653-E6-P1-CC


title: Human Lung DTC rep 1 [Seagen]


title: Rag1KO_MA_GEX_ASC1350A1

description: 10x Genomics


title: M1-Right-05-Glia-H3K27me3-R2


title: THP1, CYR61 5, rep5

city: Saint Louis

first: Joseph

middle: B

last: Lin

state: MO

address: 660 South Euclid Ave, MSC-8096-06-07

department: Ophthalmology & Visual Sciences

institute: Washington University School of Medicine

description:
all.gene_counts.tsv
group_5ug_Differential_Expression.tsv
group_0.50ug_Differential_Expression.tsv
group_0.05ug_Differential_Expression.tsv
sample20

data_processing:
Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software with a maximum of one mismatch in the indexing read. RNA-seq reads were then aligned to the Ensembl release 101 primary assembly with STAR version 2.7.9a1. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 2.0.32. Isoform expression of known Ensembl transcripts were quantified with Salmon version 1.5.23. Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 4.04.
Assembly: Ensembl release 101 primary assembly
Supplementary files format and content: all.gene_counts.tsv: Raw counts.
Supplementary files format and content: group_5ug_Differential_Expression.tsv: Differential gene expression performed comparing control vs. CYR61 5
Supplementary files format and content: group_0.50ug_Differential_Expression.tsv: Differential gene expression performed comparing control vs. CYR61 0.5
Supplementary files format and content: group_0.05ug_Differential_Expression.tsv: Differential gene expression performed comparing control vs. CYR61 0.05

source_name: cell line

growth_protocol: We used THP1 monocytes (ATCC TIB-202). These cells were maintained in RPMI 1640 media containing 1% L-glutamine, 1% penicillin/streptomycin, and 10% FBS.

extract_protocol:
After incubation, we washed cells twice with PBS, then used TRIzol (ambion) and RNeasy Plus Mini Kit (Qiagen) to extract RNA following the manufacturer's recommended protocol.
We determined total RNA integrity using an Agilent Bioanalyzer or 4200 Tapestation. Using 500 ng - 1 ug of starting total RNA, we removed ribosomal RNA using an RNase-H method using RiboErase kits (Kapa Biosystems). We fragmented mRNA in reverse transcriptase buffer and heated samples to 94 degrees for 8 minutes. We reverse transcribed mRNA to cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. We performed a second strand reaction to yield ds-cDNA. We blunt ended the cDNA, added an A base to the 3' ends, and then ligated Illumina sequencing adapters to the ends. We amplified the ligated fragments for 12-15 cycles using primers incorporating unique dual index tags. We sequenced fragments on an Illumina NovaSeq 6000 using paired end reads extending 150 bases.

treatment_protocol: We treated THP1 cells with recombinant human CYR61 (PeproTech 120-25): 0.00, 0.05, 0.50, and 5.00 μg/ml for 24 hr.

value: cell line

value: monocytes

value: CYR61, 5 ug/ml


title: Camellia-seq-lineage_LL653-E1-P3-TC_rep2

first: SHOU-WEN

address: Shilongshan ST #18, Xihu District

institute: Westlake University

value: FetalLiver-HSC


title: Rag1KO__PBS_GEX_ASC1350A2

description: 10x Genomics


title: Camellia-seq-lineage_LL653-E1-P3-TC

value: TC


title: THP1, CYR61 5, rep4

description:
all.gene_counts.tsv
group_5ug_Differential_Expression.tsv
group_0.50ug_Differential_Expression.tsv
group_0.05ug_Differential_Expression.tsv
sample19


title: M1-Right-06-Glia-H3K27me3-R1

first: Chang

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

institute: Virginia Tech


title: Human Lung DTC rep 3 [Seagen]


title: C57_PBS_TCR_ASC1350A8

description: 10x Genomics


title: M1-Right-05-Glia-H3K27me3-R1


title: M1-Right-06-Neuron-H3K27me3-R1


title: M1-Right-04-Neuron-H3K27me3-R2


title: Camellia-seq-lineage_LL653-E1-P3-RC_rep2


title: THP1, CYR61 5, rep3

description:
all.gene_counts.tsv
group_5ug_Differential_Expression.tsv
group_0.50ug_Differential_Expression.tsv
group_0.05ug_Differential_Expression.tsv
sample18


title: C57_PBS_GEX_ASC1350A4

description: 10x Genomics


title: M1-Right-04-Neuron-H3K27me3-R1


title: Camellia-seq-lineage_LL653-E1-P3-RC


title: Camellia-seq-lineage_LL653-E1-P3-CC_rep2

value: CC


title: THP1, CYR61 5, rep2

description:
all.gene_counts.tsv
group_5ug_Differential_Expression.tsv
group_0.50ug_Differential_Expression.tsv
group_0.05ug_Differential_Expression.tsv
sample17


title: THP1, CYR61 5, rep1

description:
all.gene_counts.tsv
group_5ug_Differential_Expression.tsv
group_0.50ug_Differential_Expression.tsv
group_0.05ug_Differential_Expression.tsv
sample16


title: C57_PBS_BCR_ASC1350A6

description: 10x Genomics

value: Hindlimb muscle


title: THP1, CYR61 0.5, rep5

description:
all.gene_counts.tsv
group_5ug_Differential_Expression.tsv
group_0.50ug_Differential_Expression.tsv
group_0.05ug_Differential_Expression.tsv
sample15

value: CYR61, 0.5 ug/ml


title: Camellia-seq-lineage_LL653-E1-P3-CC


title: M1-Right-04-Glia-H3K27me3-R1


title: Camellia-seq-lineage_LL653-E1-P2-TC_rep2


title: M1-Right-03-Neuron-H3K27me3-R2


title: M1-Right-03-Neuron-H3K27me3-R1


title: M1-Right-04-Glia-H3K27me3-R2


title: THP1, CYR61 0.5, rep4

description:
all.gene_counts.tsv
group_5ug_Differential_Expression.tsv
group_0.50ug_Differential_Expression.tsv
group_0.05ug_Differential_Expression.tsv
sample14


title: C57_MA_GEX_ASC1350A3

description: 10x Genomics

value: MA