title: T46_10xGenomics_3_4lines_7650_intact-cells

first: Christoph

last: Bock

address: Lazarettgasse 14

institute: CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences

description:
Fig. 2e-h, Fig. S8a-f, Fig. S9, Fig. S10, Fig. S11
Technology comparison: 10x Genomics Chromium, 7.5k intact cells per microfluidic channel

data_processing:
Library strategy: scifi-RNA-seq
Bases are called using Illumina RTA basecalling v3.4.4
Illumina basecalls are converted to undemultiplexed bam format using the IlluminaBasecallsToMultiplexSam command of Picard v2.19.2-CeMM published on the GitHub repository https://github.com/epigen/picard/releases/tag/2.19.2-CeMM
The undemultiplexed bam file is demultiplexed into single-well bam files using the IlluminaSamDemux command of Picard v2.19.2-CeMM published on the GitHub repository https://github.com/epigen/picard/releases/tag/2.19.2-CeMM
Single-well bam files are merged using samtools merge command v1.9
Single well bam files are aligned using STAR v2.7.0e and annotated using featureCounts v2.0.0 using gene features with the -Q 30 -s 0 parameters
UMI counts are quantified for each cell barcode at gene level using the scifi_pipeline.summarizer.py script published on the GitHub repository https://github.com/epigen/scifiRNA-seq_publication
Genome_build: hg38
Genome_build: mm10_e99
Supplementary_files_format_and_content: h5ad matrices with UMI counts for each cell barcode and ENSEMBL gene id for gene features
Supplementary_files_format_and_content: csv metadata files containing additional experiment information for each well-specific barcode

source_name: Human cell lines: Jurkat E6.1, HEK293T, NALM-6, K562

growth_protocol: Human Jurkat E6.1, K562, NALM-6 cells were cultured in RPMI medium supplemented with 10% FCS and penicillin/streptomycin; Human HEK293T cells were cultured in DMEM medium supplemented with 10% FCS and penicillin/streptomycin

extract_protocol:
Fresh cells were used as input for 10x Genomics profiling
Reverse Transcription: 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and obtained at 100 µM concentration in EB Buffer on 96-well plates (sequences are provided in Supplementary Table 1). 384-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at -20 ˚C (1 µl of 25 µM per well). 10,000 permeabilized cells or nuclei (2 µl of a 5,000/µl suspension) were added to the pre-dispensed primers in each well, and the assignment of samples to wells was recorded. The plate was incubated for 5 min at 55 ˚C to resolve RNA secondary structures, then placed immediately on ice to prevent their re-formation. Per well, a mix of 3 µl nuclease-free water, 2 µl 5x Reverse Transcription Buffer, 0.5 µl of 100 mM DTT (freshly diluted from Sigma #646563-10x.5ML), 0.5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 0.5 µl of RNaseOUT RNase inhibitor (40 U/ml, Thermo Fisher Scientific #10777019), and 0.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753) were added. The reverse transcription was incubated as follows (with heated lid set to 60 ˚C): 50 ˚C for 10 min, 3 cycles of [8 ˚C for 12 s, 15 ˚C for 45 s, 20 ˚C for 45 s, 30 ˚C for 30 s, 42 ˚C for 2 min, 50 ˚C for 3 min], 50 ˚C for 5 min, store at 4 ˚C.  Cell/Nuclei recovery and pooling: Processed cells/nuclei were recovered from the 384-well plate and pooled in one 15 ml tube per plate on ice. Wells were washed with ice-cold 1x PBS containing 1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 15 ml with 1x PBS containing 1% BSA, and nuclei were collected (500 rcf, 5 min, 4 ˚C). The resulting pellet was resuspended in 1.0 ml of 1x Ampligase Reaction Buffer (Lucigen #A0102K), filtered through a cell strainer (40 µm or 70 µm depending on the cell/nuclei size) into a 1.5 ml tube, and centrifuged (500 rcf, 5 min, 4 ˚C). The supernatant was removed almost completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4 ˚C) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in ~10 µl of a highly concentrated suspension, which was diluted 1:200 with 1x Ampligase Buffer and counted in a Fuchs Rosenthal counting chamber (Incyto #DHC-F01). The desired number of cells/nuclei was brought to a volume of 15 µl with 1x Ampligase Reaction Buffer (Lucigen #A0102K).  Microfluidic thermoligation barcoding: Unused channels in the Chromium Chip E (10x Genomics #2000121) were filled with 75 µl (inlet 1), 40 µl (inlet 2), and 240 µl (inlet 3) of 50% glycerol solution (Sigma #G5516-100ML). Right before loading the chip, a mix of 47.4 µl nuclease-free water, 11.5 µl of 10x Ampligase Reaction Buffer (Lucigen #A0102K), 2.3 µl of 100 U/µl Ampligase (Lucigen #A0102K), 1.5 µl of Reducing Agent B (10x Genomics #2000087), and 2.3 µl of 100 µM Bridge Oligo (sequence provided in Supplementary Table 1) was added per sample. The microfluidic chip was loaded with 75 µl of cells or nuclei in thermoligation mix (inlet 1), 40 µl of Single Cell ATAC Gel Beads (inlet 2, 10x Genomics #2000132), and 240 µl of Partitioning Oil (inlet 3, 10x Genomics #220088) and run on the Chromium system. For thermoligation barcoding, the droplet emulsion was incubated as follows (heated lid set to 105 ˚C, volume set to 100 µl): 12 cycles of [98 ˚C for 30 s, 59 ˚C for 2 min], storage at 15 ˚C. The emulsion was broken by addition of 125 µl Recovery Agent (10x Genomics #220016), and 125 µl of the pink oil phase were removed by pipetting. The remaining sample was mixed with 200 µl of Dynabead Cleanup Master Mix (per reaction: 182 µl Cleanup Buffer (10x Genomics #2000088), 8 µl Dynabeads MyOne Silane (Thermo Fisher Scientific #37002D), 5 µl Reducing Agent B (10x Genomics #2000087), 5 µl of nuclease-free water). After 10 min of incubation at room temperature, samples were washed twice with 200 µl of freshly prepared 80% ethanol (Merck #603-002-00-5) and eluted in 40.5 µl of EB Buffer (Qiagen #19086) containing 0.1% Tween (Sigma #P7949-500ML) and 1% v/v Reducing Agent B. Bead clumps were sheared with a 10 µl pipette or needle. 40 µl of the sample were transferred to a fresh tube strip and subjected to a 1.0x cleanup with SPRIselect beads (Beckman Coulter #B23318), eluting in 22 µl of EB Buffer.  Template switching: 20 µl of sample from the previous step were mixed with 10 µl of 5x Reverse Transcription Buffer, 10 µl of Ficoll PM-400 (20%, Sigma #F5415-50ML), 5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 1.25 µl of Recombinant Ribonuclease Inhibitor (Takara #2313A), 1.25 µl of 100 µM Template Switching Oligo (sequence provided in Supplementary Table 1), and 2.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753). The template switching reaction was incubated for 30 min at 25 ˚C, 90 min at 42 ˚C, storage at 4 ˚C, and cleaned with a 1.0x SPRI cleanup, eluting in 17 µl of EB buffer.  cDNA enrichment: 15 µl of the above sample were mixed with 33 µl of nuclease-free water, 50 µl of NEBNext High-Fidelity 2x PCR Master Mix (NEB #M0541S), 0.5 µl of 100 µM Partial P5 primer, 0.5 µl of 100 µM TSO Enrichment Primer (sequences provided in Supplementary Table 1), and 1 µl of 100x SYBR Green in DMSO (Life Technologies #S7563). cDNA was amplified in a thermocycler as follows: 98 ˚C for 30 s, cycle until fluorescent signal >1000 RFU [98 ˚C for 20 s, 65 ˚C for 30 s, 72 ˚C for 3 min], 72 ˚C for 5 min in another thermocycler, storage at 4 ˚C. cDNA was cleaned by one 0.8x SPRI cleanup followed by a 0.6x SPRI cleanup, quantified with a Qubit HS assay (ThermoFisher Scientific #Q32854), and 1.5 ng were checked on a Bioanalyzer High-Sensitivity DNA chip (Agilent #5067-4626 and 5067-4627).  Tagmentation: To achieve maximum library complexity, the entire sample was processed in multiple tagmentation reactions with 1 ng input each. cDNA was diluted to 0.2 ng/µl with nuclease-free water and 5 µl (1 ng) per reaction were distributed into a 96-well plate on ice. A mix of 11.25 µl nuclease-free water, 5 µl of 5x Tn5 Reaction Buffer (50 mM TAPS (Sigma #T9659-100G), 25 mM MgCl2 (Ambion #AM9530G), pH adjusted to 8.5, sterile-filtered), 2.5 µl of Dimethylformamide (Sigma #D4551-250ML), and 1.25 µl of freshly diluted i7-only transposome (prepared as described above and diluted 1:4.5 in Tn5 Dilution Buffer (50 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 100 mM NaCl (Sigma #S5150-1L), 0.1 mM EDTA (Invitrogen #AM9260G), 50% glycerol (Sigma #G5516-100ML), 0.1% Triton-X100 (Sigma #X100-100ML), 1 mM freshly added DTT (Sigma #646563-10x.5ML)) was added. Reactions were incubated for 10 min at 55 ˚C, then cooled for 1 min on ice. The enzyme was inactivated by addition of 2.5 µl of 1% SDS (Sigma #71736-100ML) for 5 min at room temperature. Next, the volume was brought to 50 µl and the fragmented cDNA was purified with a 1.0x SPRI cleanup, eluting in 17 µl of EB buffer (Qiagen #19086).  Library enrichment: 15 µl of tagmented cDNA were mixed with 5 µl of 10 µM barcoded P7 primer (sequences provided in Supplementary Table 1). Per reaction, a mix of 28.5 µl nuclease-free water, 50 µl NEBNext High-Fidelity 2x PCR Master Mix (NEB #M0541S), 1 µl of 100x SYBR Green in DMSO (Life Technologies #S7563), and 0.5 µl of 100 µM Partial-P5 primer were added. Reactions were incubated in a qPCR cycler as follows: 72 ˚C for 3 min (for end fill-in after tagmentation), 98 ˚C for 30 s, cycle [98 ˚C for 10 s, 65 ˚C for 30 s, 72 ˚C for 30 s, plate read]. We monitored the fluorescence signals and removed samples from the thermocycler when they reached >4000 RFU. To complete unfinished PCR products, we incubated for 5 min at 72 ˚C in another thermocycler. Libraries were cleaned with a 0.7x SPRI cleanup with AMPure XP beads. All wells with the same P7 barcode were po

treatment_protocol: No treatments were performed

tag: cell line

value: Jurkat E6.1, HEK293T, NALM-6, K562

tag: annotation round1 barcodes

value: No round1 barcoding


title: SE21s_ZT18

status: Public on Mar 11 2021

type: SRA

city: Beijing

first: Zhen

last: Zhu

address: No.1 West Beichen Road, Chaoyang District

country: China

institute: Institute of Genetics and Developmental Biology

description:
mRNA profiling
AllSamples_FPKM.txt

library_source: transcriptomic

data_processing:
Illumina GA Pipeline version 1.5 was used for base-calling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then filtered into clean reads.
The expression levels of each gene were quantified by calculated expected number of fragments per kilobase of transcript per millions fragments sequenced (FPKM) using Cuffdiff 2
The mRNA-Seq clean reads were aligned to the gene models in MSU Rice Genome Annotation release 7.0 using TopHat 2.
The gene expression level was calculated by using fragments per kilobase transcriptome per million mapped reads (FPKM) method, with unique position matched reads.
Genome_build: MSU Rice Genome Annotation release 7.0
Supplementary_files_format_and_content: Tab-delimited text files include FPKM values for each Sample.

molecule: total RNA

organism: Oryza sativa

source_name: Flag leaves

growth_protocol: Super-hybrid rice LY2186 and its parental lines, sterile line SE21s and restorer line MH86 were planted in the field under the same environmental conditions. Flag leaf  samples were collected at grain-filling stage during 17 serial time points (circadian clock ZT0~ZT48, 3h interval). The samples were collected, frozen in liquid nitrogen and stored at –80 °C for total RNA extraction.

extract_protocol:
Total RNAs were extracted from the collected flag leaf at grain-filling stage of hybrid rice and its parents using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol.
Strand-specific transcriptome libraries were constructed using TruSeq® Stranded RNA LT Kit (Illumina, San Diego, CA, USA) by following the manufacturer’s instructions.

tag: strain

tag: tissue

value: Flag leaves

tag: developmental stage

value: Grain-filling stage

tag: timepoint


title: T46_10xGenomics_2_4lines_7650_MeOH-cells

city: Vienna

country: Austria

description:
Fig. 2e-h, Fig. S8a-f, Fig. S9, Fig. S10, Fig. S11
Technology comparison: 10x Genomics Chromium, 7.5k methanol-fixed cells per microfluidic channel

molecule: polyA RNA

organism: Homo sapiens

extract_protocol:
Methanol fixation of cells: A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifuga-tion (300 rcf, 5 min, 4 ˚C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific #M/4000/17) at -20 ˚C for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4 ˚C) with 5 ml of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA (Sigma #A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) permeabilized cells were resuspended in 200 µl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 cells per µl with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq.
Reverse Transcription: 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and obtained at 100 µM concentration in EB Buffer on 96-well plates (sequences are provided in Supplementary Table 1). 384-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at -20 ˚C (1 µl of 25 µM per well). 10,000 permeabilized cells or nuclei (2 µl of a 5,000/µl suspension) were added to the pre-dispensed primers in each well, and the assignment of samples to wells was recorded. The plate was incubated for 5 min at 55 ˚C to resolve RNA secondary structures, then placed immediately on ice to prevent their re-formation. Per well, a mix of 3 µl nuclease-free water, 2 µl 5x Reverse Transcription Buffer, 0.5 µl of 100 mM DTT (freshly diluted from Sigma #646563-10x.5ML), 0.5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 0.5 µl of RNaseOUT RNase inhibitor (40 U/ml, Thermo Fisher Scientific #10777019), and 0.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753) were added. The reverse transcription was incubated as follows (with heated lid set to 60 ˚C): 50 ˚C for 10 min, 3 cycles of [8 ˚C for 12 s, 15 ˚C for 45 s, 20 ˚C for 45 s, 30 ˚C for 30 s, 42 ˚C for 2 min, 50 ˚C for 3 min], 50 ˚C for 5 min, store at 4 ˚C.  Cell/Nuclei recovery and pooling: Processed cells/nuclei were recovered from the 384-well plate and pooled in one 15 ml tube per plate on ice. Wells were washed with ice-cold 1x PBS containing 1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 15 ml with 1x PBS containing 1% BSA, and nuclei were collected (500 rcf, 5 min, 4 ˚C). The resulting pellet was resuspended in 1.0 ml of 1x Ampligase Reaction Buffer (Lucigen #A0102K), filtered through a cell strainer (40 µm or 70 µm depending on the cell/nuclei size) into a 1.5 ml tube, and centrifuged (500 rcf, 5 min, 4 ˚C). The supernatant was removed almost completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4 ˚C) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in ~10 µl of a highly concentrated suspension, which was diluted 1:200 with 1x Ampligase Buffer and counted in a Fuchs Rosenthal counting chamber (Incyto #DHC-F01). The desired number of cells/nuclei was brought to a volume of 15 µl with 1x Ampligase Reaction Buffer (Lucigen #A0102K).  Microfluidic thermoligation barcoding: Unused channels in the Chromium Chip E (10x Genomics #2000121) were filled with 75 µl (inlet 1), 40 µl (inlet 2), and 240 µl (inlet 3) of 50% glycerol solution (Sigma #G5516-100ML). Right before loading the chip, a mix of 47.4 µl nuclease-free water, 11.5 µl of 10x Ampligase Reaction Buffer (Lucigen #A0102K), 2.3 µl of 100 U/µl Ampligase (Lucigen #A0102K), 1.5 µl of Reducing Agent B (10x Genomics #2000087), and 2.3 µl of 100 µM Bridge Oligo (sequence provided in Supplementary Table 1) was added per sample. The microfluidic chip was loaded with 75 µl of cells or nuclei in thermoligation mix (inlet 1), 40 µl of Single Cell ATAC Gel Beads (inlet 2, 10x Genomics #2000132), and 240 µl of Partitioning Oil (inlet 3, 10x Genomics #220088) and run on the Chromium system. For thermoligation barcoding, the droplet emulsion was incubated as follows (heated lid set to 105 ˚C, volume set to 100 µl): 12 cycles of [98 ˚C for 30 s, 59 ˚C for 2 min], storage at 15 ˚C. The emulsion was broken by addition of 125 µl Recovery Agent (10x Genomics #220016), and 125 µl of the pink oil phase were removed by pipetting. The remaining sample was mixed with 200 µl of Dynabead Cleanup Master Mix (per reaction: 182 µl Cleanup Buffer (10x Genomics #2000088), 8 µl Dynabeads MyOne Silane (Thermo Fisher Scientific #37002D), 5 µl Reducing Agent B (10x Genomics #2000087), 5 µl of nuclease-free water). After 10 min of incubation at room temperature, samples were washed twice with 200 µl of freshly prepared 80% ethanol (Merck #603-002-00-5) and eluted in 40.5 µl of EB Buffer (Qiagen #19086) containing 0.1% Tween (Sigma #P7949-500ML) and 1% v/v Reducing Agent B. Bead clumps were sheared with a 10 µl pipette or needle. 40 µl of the sample were transferred to a fresh tube strip and subjected to a 1.0x cleanup with SPRIselect beads (Beckman Coulter #B23318), eluting in 22 µl of EB Buffer.  Template switching: 20 µl of sample from the previous step were mixed with 10 µl of 5x Reverse Transcription Buffer, 10 µl of Ficoll PM-400 (20%, Sigma #F5415-50ML), 5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 1.25 µl of Recombinant Ribonuclease Inhibitor (Takara #2313A), 1.25 µl of 100 µM Template Switching Oligo (sequence provided in Supplementary Table 1), and 2.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753). The template switching reaction was incubated for 30 min at 25 ˚C, 90 min at 42 ˚C, storage at 4 ˚C, and cleaned with a 1.0x SPRI cleanup, eluting in 17 µl of EB buffer.  cDNA enrichment: 15 µl of the above sample were mixed with 33 µl of nuclease-free water, 50 µl of NEBNext High-Fidelity 2x PCR Master Mix (NEB #M0541S), 0.5 µl of 100 µM Partial P5 primer, 0.5 µl of 100 µM TSO Enrichment Primer (sequences provided in Supplementary Table 1), and 1 µl of 100x SYBR Green in DMSO (Life Technologies #S7563). cDNA was amplified in a thermocycler as follows: 98 ˚C for 30 s, cycle until fluorescent signal >1000 RFU [98 ˚C for 20 s, 65 ˚C for 30 s, 72 ˚C for 3 min], 72 ˚C for 5 min in another thermocycler, storage at 4 ˚C. cDNA was cleaned by one 0.8x SPRI cleanup followed by a 0.6x SPRI cleanup, quantified with a Qubit HS assay (ThermoFisher Scientific #Q32854), and 1.5 ng were checked on a Bioanalyzer High-Sensitivity DNA chip (Agilent #5067-4626 and 5067-4627).  Tagmentation: To achieve maximum library complexity, the entire sample was processed in multiple tagmentation reactions with 1 ng input each. cDNA was diluted to 0.2 ng/µl with nuclease-free water and 5 µl (1 ng) per reaction were distributed into a 96-well plate on ice. A mix of 11.25 µl nuclease-free water, 5 µl of 5x Tn5 Reaction Buffer (50 mM TAPS (Sigma #T9659-100G), 25 mM MgCl2 (Ambion #AM9530G), pH adjusted to 8.5, sterile-filtered), 2.5 µl of Dimethylformamide (Sigma #D4551-250ML), and 1.25 µl of freshly diluted i7-only transposome (prepared as described above and diluted 1:4.5 in Tn5 Dilution Buffer (50 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 100 mM NaCl (Sigma #S5150-1L), 0.1 mM EDTA (Invitrogen #AM9260G), 50% glycerol (Sigma #G5516-100ML), 0.1% Triton-X100 (Sigma #X100-100ML), 1 mM freshly added DTT (Sigma #646563-10x.5ML)) was added. Reactions were incubated for 10 min at 55 ˚C, then cooled for 1 min on ice. The enzyme was inactivated by addition of 2.5 µl of 1% SDS (Sigma #71736-100ML) for 5 min at room temperature. Next, the volume was brought to 50 µl and the fragmented cDNA was purified with a 1.0x SPRI cleanup, eluting in 17 µl of EB buffer (Qiagen #19086).  Library enrichment: 15 µl of tagmented cDNA


title: SE21s_ZT15

description:
mRNA profiling
AllSamples_FPKM.txt


title: PD209_S_scATAC-std_libprep-std

description:
Fig. S7
scifi-RNA-seq on the scATAC v1.0 platform using cells or nuclei, and either Superscript IV or Maxima H Minus as reverse transcriptase

organism:
Homo sapiens
Mus musculus

source_name: Human cell line Jurkat E6.1, Mouse cell line 3T3

growth_protocol: Human Jurkat E6.1 cells were cultured in RPMI medium supplemented with 10% FCS and penicillin/streptomycin; Mouse 3T3 cells were cultured in DMEM medium supplemented with 10% FCS and penicillin/streptomycin

extract_protocol:
Fresh nuclei isolation: A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 ˚C). Nuclei were prepared by resuspending cells in 500 µl of ice-cold Nuclei Preparation Buffer (10 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 10 mM NaCl (Sigma #S5150-1L), 3 mM MgCl2 (Ambion #AM9530G), 1% w/v BSA (Sigma #A8806-5), 1% v/v SUPERase-In RNase Inhibitor (20 U/µl, Thermo Fisher Scientific #AM2696), 0.1% v/v Tween-20 (Sigma #P7949-500ML), 0.1% v/v IGEPAL CA-630 (Sigma #I8896-50ML), 0.01% v/v Digitonin (Promega #G944A)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by adding 5 ml of ice-cold Nuclei Wash Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.1% v/v Tween-20). Nuclei were collected by centrifugation (500 rcf, 5 min, 4 ˚C), resuspended in 200 µl of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA and 1% v/v SUPERase-In RNase Inhibi-tor) and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 nuclei per µl with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq. Methanol fixation of cells: A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifuga-tion (300 rcf, 5 min, 4 ˚C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific #M/4000/17) at -20 ˚C for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4 ˚C) with 5 ml of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA (Sigma #A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) permeabilized cells were resuspended in 200 µl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 cells per µl with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq.
Reverse Transcription: 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and obtained at 100 µM concentration in EB Buffer on 96-well plates (sequences are provided in Supplementary Table 1). 384-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at -20 ˚C (1 µl of 25 µM per well). 10,000 permeabilized cells or nuclei (2 µl of a 5,000/µl suspension) were added to the pre-dispensed primers in each well, and the assignment of samples to wells was recorded. The plate was incubated for 5 min at 55 ˚C to resolve RNA secondary structures, then placed immediately on ice to prevent their re-formation. Per well, a mix of 3 µl nuclease-free water, 2 µl 5x Reverse Transcription Buffer, 0.5 µl of 100 mM DTT (freshly diluted from Sigma #646563-10x.5ML), 0.5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 0.5 µl of RNaseOUT RNase inhibitor (40 U/ml, Thermo Fisher Scientific #10777019), and 0.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753) were added. The reverse transcription was incubated as follows (with heated lid set to 60 ˚C): 50 ˚C for 10 min, 3 cycles of [8 ˚C for 12 s, 15 ˚C for 45 s, 20 ˚C for 45 s, 30 ˚C for 30 s, 42 ˚C for 2 min, 50 ˚C for 3 min], 50 ˚C for 5 min, store at 4 ˚C.  Cell/Nuclei recovery and pooling: Processed cells/nuclei were recovered from the 384-well plate and pooled in one 15 ml tube per plate on ice. Wells were washed with ice-cold 1x PBS containing 1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 15 ml with 1x PBS containing 1% BSA, and nuclei were collected (500 rcf, 5 min, 4 ˚C). The resulting pellet was resuspended in 1.0 ml of 1x Ampligase Reaction Buffer (Lucigen #A0102K), filtered through a cell strainer (40 µm or 70 µm depending on the cell/nuclei size) into a 1.5 ml tube, and centrifuged (500 rcf, 5 min, 4 ˚C). The supernatant was removed almost completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4 ˚C) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in ~10 µl of a highly concentrated suspension, which was diluted 1:200 with 1x Ampligase Buffer and counted in a Fuchs Rosenthal counting chamber (Incyto #DHC-F01). The desired number of cells/nuclei was brought to a volume of 15 µl with 1x Ampligase Reaction Buffer (Lucigen #A0102K).  Microfluidic thermoligation barcoding: Unused channels in the Chromium Chip E (10x Genomics #2000121) were filled with 75 µl (inlet 1), 40 µl (inlet 2), and 240 µl (inlet 3) of 50% glycerol solution (Sigma #G5516-100ML). Right before loading the chip, a mix of 47.4 µl nuclease-free water, 11.5 µl of 10x Ampligase Reaction Buffer (Lucigen #A0102K), 2.3 µl of 100 U/µl Ampligase (Lucigen #A0102K), 1.5 µl of Reducing Agent B (10x Genomics #2000087), and 2.3 µl of 100 µM Bridge Oligo (sequence provided in Supplementary Table 1) was added per sample. The microfluidic chip was loaded with 75 µl of cells or nuclei in thermoligation mix (inlet 1), 40 µl of Single Cell ATAC Gel Beads (inlet 2, 10x Genomics #2000132), and 240 µl of Partitioning Oil (inlet 3, 10x Genomics #220088) and run on the Chromium system. For thermoligation barcoding, the droplet emulsion was incubated as follows (heated lid set to 105 ˚C, volume set to 100 µl): 12 cycles of [98 ˚C for 30 s, 59 ˚C for 2 min], storage at 15 ˚C. The emulsion was broken by addition of 125 µl Recovery Agent (10x Genomics #220016), and 125 µl of the pink oil phase were removed by pipetting. The remaining sample was mixed with 200 µl of Dynabead Cleanup Master Mix (per reaction: 182 µl Cleanup Buffer (10x Genomics #2000088), 8 µl Dynabeads MyOne Silane (Thermo Fisher Scientific #37002D), 5 µl Reducing Agent B (10x Genomics #2000087), 5 µl of nuclease-free water). After 10 min of incubation at room temperature, samples were washed twice with 200 µl of freshly prepared 80% ethanol (Merck #603-002-00-5) and eluted in 40.5 µl of EB Buffer (Qiagen #19086) containing 0.1% Tween (Sigma #P7949-500ML) and 1% v/v Reducing Agent B. Bead clumps were sheared with a 10 µl pipette or needle. 40 µl of the sample were transferred to a fresh tube strip and subjected to a 1.0x cleanup with SPRIselect beads (Beckman Coulter #B23318), eluting in 22 µl of EB Buffer.  Template switching: 20 µl of sample from the previous step were mixed with 10 µl of 5x Reverse Transcription Buffer, 10 µl of Ficoll PM-400 (20%, Sigma #F5415-50ML), 5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 1.25 µl of Recombinant Ribonuclease Inhibitor (Takara #2313A), 1.25 µl of 100 µM Template Switching Oligo (sequence provided in Supplementary Table 1), and 2.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753). The template switching reaction was incubated for 30 min at 25 ˚C, 90 min at 42 ˚C, storage at 4 ˚C, and cleaned with a 1.0x SPRI cleanup, eluting in 17 µl of EB buffer.  cDNA enrichment: 15 µl of the above sample were mixed with 33 µl of nuclease-free water, 50 µl of NEBNext High-Fidelity 2x PCR Master Mix (NEB #M0541S), 0.5 µl of 100 µM Partial P5 primer, 0.5 µl of 100 µM TSO Enrichment Primer (sequences provided in Supplementary Table 1), and 1 µl of 100x SYBR Green in DMSO (Life Technologies #S7563). cDNA was amplified in a thermocycler as follows: 98 ˚C for 30 s, cycle until fluorescent signal >1000 RFU [98 ˚C for 20 s, 65 ˚C for 30 s, 72 ˚C for 3 min], 72 ˚C for 5 min in another thermocycler, storage at 4 ˚C. cDNA was cleaned by one 0.8x SPRI cleanup followed by a 0.6x SPRI cleanup, quantified with a Qubit HS assay (ThermoFisher Scientific #Q32854)

value: Jurkat E6.1, Mouse cell line 3T3


title: T46_10xGenomics_1_4lines_7650_nuclei

description:
Fig. 2e-h, Fig. S8a-f, Fig. S9, Fig. S10, Fig. S11
Technology comparison: 10x Genomics Chromium, 7.5k nuclei per microfluidic channel

extract_protocol:
Fresh nuclei isolation: A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 ˚C). Nuclei were prepared by resuspending cells in 500 µl of ice-cold Nuclei Preparation Buffer (10 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 10 mM NaCl (Sigma #S5150-1L), 3 mM MgCl2 (Ambion #AM9530G), 1% w/v BSA (Sigma #A8806-5), 1% v/v SUPERase-In RNase Inhibitor (20 U/µl, Thermo Fisher Scientific #AM2696), 0.1% v/v Tween-20 (Sigma #P7949-500ML), 0.1% v/v IGEPAL CA-630 (Sigma #I8896-50ML), 0.01% v/v Digitonin (Promega #G944A)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by adding 5 ml of ice-cold Nuclei Wash Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.1% v/v Tween-20). Nuclei were collected by centrifugation (500 rcf, 5 min, 4 ˚C), resuspended in 200 µl of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA and 1% v/v SUPERase-In RNase Inhibi-tor) and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 nuclei per µl with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq.
Reverse Transcription: 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and obtained at 100 µM concentration in EB Buffer on 96-well plates (sequences are provided in Supplementary Table 1). 384-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at -20 ˚C (1 µl of 25 µM per well). 10,000 permeabilized cells or nuclei (2 µl of a 5,000/µl suspension) were added to the pre-dispensed primers in each well, and the assignment of samples to wells was recorded. The plate was incubated for 5 min at 55 ˚C to resolve RNA secondary structures, then placed immediately on ice to prevent their re-formation. Per well, a mix of 3 µl nuclease-free water, 2 µl 5x Reverse Transcription Buffer, 0.5 µl of 100 mM DTT (freshly diluted from Sigma #646563-10x.5ML), 0.5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 0.5 µl of RNaseOUT RNase inhibitor (40 U/ml, Thermo Fisher Scientific #10777019), and 0.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753) were added. The reverse transcription was incubated as follows (with heated lid set to 60 ˚C): 50 ˚C for 10 min, 3 cycles of [8 ˚C for 12 s, 15 ˚C for 45 s, 20 ˚C for 45 s, 30 ˚C for 30 s, 42 ˚C for 2 min, 50 ˚C for 3 min], 50 ˚C for 5 min, store at 4 ˚C.  Cell/Nuclei recovery and pooling: Processed cells/nuclei were recovered from the 384-well plate and pooled in one 15 ml tube per plate on ice. Wells were washed with ice-cold 1x PBS containing 1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 15 ml with 1x PBS containing 1% BSA, and nuclei were collected (500 rcf, 5 min, 4 ˚C). The resulting pellet was resuspended in 1.0 ml of 1x Ampligase Reaction Buffer (Lucigen #A0102K), filtered through a cell strainer (40 µm or 70 µm depending on the cell/nuclei size) into a 1.5 ml tube, and centrifuged (500 rcf, 5 min, 4 ˚C). The supernatant was removed almost completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4 ˚C) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in ~10 µl of a highly concentrated suspension, which was diluted 1:200 with 1x Ampligase Buffer and counted in a Fuchs Rosenthal counting chamber (Incyto #DHC-F01). The desired number of cells/nuclei was brought to a volume of 15 µl with 1x Ampligase Reaction Buffer (Lucigen #A0102K).  Microfluidic thermoligation barcoding: Unused channels in the Chromium Chip E (10x Genomics #2000121) were filled with 75 µl (inlet 1), 40 µl (inlet 2), and 240 µl (inlet 3) of 50% glycerol solution (Sigma #G5516-100ML). Right before loading the chip, a mix of 47.4 µl nuclease-free water, 11.5 µl of 10x Ampligase Reaction Buffer (Lucigen #A0102K), 2.3 µl of 100 U/µl Ampligase (Lucigen #A0102K), 1.5 µl of Reducing Agent B (10x Genomics #2000087), and 2.3 µl of 100 µM Bridge Oligo (sequence provided in Supplementary Table 1) was added per sample. The microfluidic chip was loaded with 75 µl of cells or nuclei in thermoligation mix (inlet 1), 40 µl of Single Cell ATAC Gel Beads (inlet 2, 10x Genomics #2000132), and 240 µl of Partitioning Oil (inlet 3, 10x Genomics #220088) and run on the Chromium system. For thermoligation barcoding, the droplet emulsion was incubated as follows (heated lid set to 105 ˚C, volume set to 100 µl): 12 cycles of [98 ˚C for 30 s, 59 ˚C for 2 min], storage at 15 ˚C. The emulsion was broken by addition of 125 µl Recovery Agent (10x Genomics #220016), and 125 µl of the pink oil phase were removed by pipetting. The remaining sample was mixed with 200 µl of Dynabead Cleanup Master Mix (per reaction: 182 µl Cleanup Buffer (10x Genomics #2000088), 8 µl Dynabeads MyOne Silane (Thermo Fisher Scientific #37002D), 5 µl Reducing Agent B (10x Genomics #2000087), 5 µl of nuclease-free water). After 10 min of incubation at room temperature, samples were washed twice with 200 µl of freshly prepared 80% ethanol (Merck #603-002-00-5) and eluted in 40.5 µl of EB Buffer (Qiagen #19086) containing 0.1% Tween (Sigma #P7949-500ML) and 1% v/v Reducing Agent B. Bead clumps were sheared with a 10 µl pipette or needle. 40 µl of the sample were transferred to a fresh tube strip and subjected to a 1.0x cleanup with SPRIselect beads (Beckman Coulter #B23318), eluting in 22 µl of EB Buffer.  Template switching: 20 µl of sample from the previous step were mixed with 10 µl of 5x Reverse Transcription Buffer, 10 µl of Ficoll PM-400 (20%, Sigma #F5415-50ML), 5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 1.25 µl of Recombinant Ribonuclease Inhibitor (Takara #2313A), 1.25 µl of 100 µM Template Switching Oligo (sequence provided in Supplementary Table 1), and 2.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753). The template switching reaction was incubated for 30 min at 25 ˚C, 90 min at 42 ˚C, storage at 4 ˚C, and cleaned with a 1.0x SPRI cleanup, eluting in 17 µl of EB buffer.  cDNA enrichment: 15 µl of the above sample were mixed with 33 µl of nuclease-free water, 50 µl of NEBNext High-Fidelity 2x PCR Master Mix (NEB #M0541S), 0.5 µl of 100 µM Partial P5 primer, 0.5 µl of 100 µM TSO Enrichment Primer (sequences provided in Supplementary Table 1), and 1 µl of 100x SYBR Green in DMSO (Life Technologies #S7563). cDNA was amplified in a thermocycler as follows: 98 ˚C for 30 s, cycle until fluorescent signal >1000 RFU [98 ˚C for 20 s, 65 ˚C for 30 s, 72 ˚C for 3 min], 72 ˚C for 5 min in another thermocycler, storage at 4 ˚C. cDNA was cleaned by one 0.8x SPRI cleanup followed by a 0.6x SPRI cleanup, quantified with a Qubit HS assay (ThermoFisher Scientific #Q32854), and 1.5 ng were checked on a Bioanalyzer High-Sensitivity DNA chip (Agilent #5067-4626 and 5067-4627).  Tagmentation: To achieve maximum library complexity, the entire sample was processed in multiple tagmentation reactions with 1 ng input each. cDNA was diluted to 0.2 ng/µl with nuclease-free water and 5 µl (1 ng) per reaction were distributed into a 96-well plate on ice. A mix of 11.25 µl nuclease-free water, 5 µl of 5x Tn5 Reaction Buffer (50 mM TAPS (Sigma #T9659-100G), 25 mM MgCl2 (Ambion #AM9530G), pH adjusted to 8.5, sterile-filtered), 2.5 µl of Dimethylformamide (Sigma #D4551-250ML), and 1.25 µl of freshly diluted i7-only transposome (prepared as described above and diluted 1:4.5 in Tn5 Dilution Buffer (50 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 100 mM NaCl (Sigma #S5150-1L), 0.1 mM EDTA (Invitrogen #AM9260G), 50% glycerol (Sigma #G5516-100ML), 0.1% Triton-X100 (Sigma


title: SE21s_ZT12

description:
mRNA profiling
AllSamples_FPKM.txt


title: 5xFAD 2

city: Saint Louis

first: Jonathan

last: Kipnis

state: MO

address: 4515 McKinley Ave

department: Pathology and Immunology

country: USA

institute: Washington University in Saint Louis

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_wt_vs_5xfad_norm_counts (SL357717)

data_processing:
Raw FASTQ sequencing reads were chastity filtered to remove clusters having outlying intensity corresponding to bases other than the called base.
Filtered reads were assessed for quality using FastQC.
Filtered reads were trimmed using Trimmomatic to remove adapter sequences and low quality bases
Transcript-level abundances were calculated using Salmon.
Transcript-level abundances were summarized into gene-level counts with tximport.
Raw counts were then filtered to remove genes without at least  5 counts in at least 3 samples,  as well as genes with standard deviations of log transformed counts greater than 2 between replicates
The filtered raw counts were then normalized with DEseq2
Genome_build: mm10
Supplementary_files_format_and_content: processed data files are csv files containing gene x count matrices of the DEseq2 normalized counts.

organism: Mus musculus

source_name: Mouse brain meninges

extract_protocol:
RNA was extracted using Arcturus PicoPure kit (Thermo Fisher)
All RNA sample processing (including linear RNA amplification and cDNA library generation) and RNA-seq was performed by HudsonAlpha Genomic Services Laboratory

tag: genotype/variation

value: 5xFAD hemizygous

tag: celltype

value: meningeal lymphatic endothelial cells

tag: age

value: 6 months

tag: Sex

value: male


title: PD213_scifi_2_CRISPR-TCR_77300_MeOH-cells

description:
Fig. 3, Fig. S14
Single-cell CRISPR screen for T cell receptor signaling in Jurkat cells

source_name: Human cell line Jurkat E6.1, transduced to express SpCas9 (Addgene #52962), transduced with single CRISPR guide RNA constructs cloned into CROPseq-Guide-Puro (Addgene #86708)

growth_protocol: Human Jurkat E6.1 cells were cultured in RPMI medium supplemented with 10% FCS and penicillin/streptomycin

treatment_protocol: Human Jurkat cells (clone E6-1) were transduced with LentiCas9-Blast (Addgene #52962) and selected with 25 µg/ml of blasticidin (Invivogen #ant-bl-5) to achieve a stable expression of Cas9. Jurkat-Cas9 cells were then transduced with 48 single guide RNA constructs in an arrayed fashion: Jurkat-Cas9 cells were seeded onto a 96-well plate at 250,000 cells per well in 100 µl of complete culture medium (RPMI (Gibco #21875-034), 10% FCS (Sigma), penicillin-streptomycin) containing 25 µg/ml blasticidin to maintain the selection pressure for Cas9 and 8 µg/ml polybrene (Sigma Aldrich #H9268-5G) to facilitate the interaction of viral particles with the cells. Afterwards, each well was transduced with 10 µl of lentiviral particles and we performed spinfection at 37 ˚C and 1,200 RCF for 45 min. Following overnight incubation at 37 ˚C and 5% CO2, the medium was exchanged to select for the guide RNA construct (RPMI, 10% FCS, penicillin–streptomycin, 25 µg/ml blasticidin, 2 µg/ml puromycin (ThermoFisher Scientific #A1113803). The selective medium was renewed every 2-3 days for a duration of 10 days to allow for effi-cient genome editing, while maintaining the cells in a 96-well plate. The knockout cells were split into two equal parts while transferring to a 96-well V-bottom plate, pelleted by centrifugation at 300 RCF for 5 min, and washed once with 200 µl of starvation medium (RPMI (Gibco #21875-034), 0% FCS, penicillin-streptomycin). Cell pellets were resuspended in 200 µl of starvation medium, and serum-starved for 4 h. For T cell receptor activation, we coated one half of a flat-bottom 96-well plate with 50 µl per well of a 10 µg/ml solution of anti-human CD3 antibody in 1x PBS (clone OKT3, ThermoFisher Scientific #16-0037-81). The other half of the wells was left untreated, adding only 50 µl of 1x PBS. After 2 h of surface coating at 37 ˚C, the antibody solution was removed, and the coated plate was washed two times with 200 µl of 1x PBS. Per well, 180 µl of cells in serum-starvation medium were added, followed by either 20 µl of a 20 µg/ml solution of anti-human CD28 antibody diluted in starvation medium for stimulated wells (clone CD28.2, ThermoFisher Scientific #16-0289-81), or 20 µl of starvation medium for unstimulated cells. Cells were incubated for 4 h to allow for efficient TCR activation in antibody-treated wells.

value: Jurkat E6.1


title: 5xFAD 1

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_wt_vs_5xfad_norm_counts (SL357716)


title: PD196-2_153k_human-mouse_freshnuclei_full-protocol

description:
Fig. 2a-b
Human (Jurkat) / Mouse (3T3) mixed 1:1, 15.3k nuclei per microfluidic channel


title: Es_M_R1_1h

city: Wuhan

first: Kenichi

last: Tsuda

address: No.1 Shizishan Road

institute: Huazhong Agricultural University

description: processed data file: merged_counts_Esa.txt

data_processing:
Illumina CASAVA-1.8.2 software was used for basecalling and Illumina adapter matching reads were removed using cutadapt 1.5
Reads were mapped to A. thaliana (TAIR10), C. hirsuta (http://chi.mpipz.mpg.de/), C. rubella (Phytozome 10), E. salsugineum (Phytozome10) or individual A. thalianna accession genomes corrected for SNPs usingTopHat2 (v2.1.1) with following parameters: --read-mismatches 10 --read-gap-length 10 --read-edit-dist 20 --read-realign-edit-dist 0  --min-anchor 5 --splice-mismatches 2 --min-intron-length 30 --max-intron-length 10000 --max-insertion-length 20 --max-deletion-length 20 --num-threads 10 --max-multihits 10 --coverage-search --library-type fr-firststrand --segment-mismatches 3 --min-segment-intron 30 --max-segment-intron 10000 --min-coverage-intron 30 --max-coverage-intron 10000 --b2-very-sensitive
reads per gene were counted using HtSeq (v 0.6.0) with default parameters
Supplementary_files_format_and_content: Tab-delimited text file including raw read counts per gene for each sample.

organism: Eutrema salsugineum

source_name: Eutrema salsugineum Shandong

extract_protocol:
seedlings were flash frozen in liquid nitrogen and RNA was extracted using peqGOLD TriFast reagent
library preparation was performed with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs)

treatment_protocol: Eleven-day-old seedlings were transferred from ½ MS-Agar to 24-well plates each with 1.6 ml of ½ MS-Medium 24 h prior to treatments. Five to ten seedlings were transferred per well. For the flg22 treatment 800 µl of 3 µM flg22 solution was added to the medium containing the seedlings resulting in a final concentration of 1 µM flg22. For mock treatments 800 µl 1/2 MS medium was added to the wells. Three well were combined to create one sample.

value: seedling

value: 12 day old

tag: treatment

value: mock

tag: replicate


title: SE21s_ZT9

description:
mRNA profiling
AllSamples_FPKM.txt


title: SE21s_ZT6

description:
mRNA profiling
AllSamples_FPKM.txt


title: WT 3

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_wt_vs_5xfad_norm_counts (SL357715)

value: non-transgenic


title: PD196-1_cells-vs-nuclei-vs-fixednuclei

description:
Fig. S5, Fig. S6
Comparison of methanol-fixed cells, nuclei, nuclei fixed with 1% formaldehyde and nuclei fixed with 4% formaldehyde

extract_protocol:
Fresh nuclei isolation: A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 ˚C). Nuclei were prepared by resuspending cells in 500 µl of ice-cold Nuclei Preparation Buffer (10 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 10 mM NaCl (Sigma #S5150-1L), 3 mM MgCl2 (Ambion #AM9530G), 1% w/v BSA (Sigma #A8806-5), 1% v/v SUPERase-In RNase Inhibitor (20 U/µl, Thermo Fisher Scientific #AM2696), 0.1% v/v Tween-20 (Sigma #P7949-500ML), 0.1% v/v IGEPAL CA-630 (Sigma #I8896-50ML), 0.01% v/v Digitonin (Promega #G944A)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by adding 5 ml of ice-cold Nuclei Wash Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.1% v/v Tween-20). Nuclei were collected by centrifugation (500 rcf, 5 min, 4 ˚C), resuspended in 200 µl of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA and 1% v/v SUPERase-In RNase Inhibi-tor) and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 nuclei per µl with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq. Methanol fixation of cells: A total of 5 million cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifuga-tion (300 rcf, 5 min, 4 ˚C) and fixed in 5 ml of ice-cold methanol (Fisher Scientific #M/4000/17) at -20 ˚C for 10 min. After two additional washes (centrifugation: 300 rcf, 5 min, 4 ˚C) with 5 ml of ice-cold PBS-BSA-SUPERase (1x PBS supplemented with 1% w/v BSA (Sigma #A8806-5) and 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696)) permeabilized cells were resuspended in 200 µl of ice-cold PBS-BSA-SUPERase, and filtered through a cell strainer (40 µM or 70 µM depending on the cell size). We then used 5 µl of the sample for cell counting in duplicates on a CASY device (Schärfe System) and diluted to 5,000 cells per µl with ice-cold PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq. Fixed nuclei suspension: A total of 5 million primary cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 ˚C). Nuclei were prepared by resuspending cells in 500 µl of ice-cold Nu-clei Preparation Buffer without Digitonin and without Tween-20 (10 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 10 mM NaCl (Sigma #S5150-1L), 3 mM MgCl2 (Ambion #AM9530G), 1% w/v BSA (Sigma #A8806-5), 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696), 0.1% v/v IGEPAL CA-630 (Sigma #I8896-50ML)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by addition of 5 ml of Nuclei Wash Buffer without Tween-20 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor). Nuclei were collected by centrifugation (500 rcf, 5 min, 4 ˚C), and fixed in 5 ml of ice-cold 1x PBS containing 1% or 4% Formaldehyde (Thermo Fisher Scientific #28908) for 15 min on ice. Fixed nuclei were collected (500 rcf, 5 min, 4 ˚C), the pellet was resuspended in 1.5 ml of ice-cold Nuclei Wash Buffer without Tween-20 and transferred to a 1.5 ml tube. After one more wash with 1.5 ml of ice-cold Nuclei Wash Buffer without Tween-20 (500 rcf, 5 min, 4 ˚C), fixed nuclei were resuspended in 200 µl of Nuclei Wash Buffer without Tween-20, snap-frozen in liquid nitrogen and stored at -80 ˚C.  For scifi-RNA-seq, frozen samples were thawed in a 37 ˚C water bath for exactly 1 min, and immediately placed on ice. Following centrifugation (500 rcf, 5 min, 4 ˚C), fixed nuclei were resuspended in 250 µl of ice-cold Permeabilization Buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.01% v/v Digitonin (Promega #G944A), 0.1% v/v Tween-20 (Sigma #P7949-500ML)). After 5 min of incubation on ice, 250 µl of Nuclei Wash Buffer without Tween-20 were added per sample, and nuclei were collected (500 rcf, 5 min, 4 ˚C). After one more wash with 250 µl of Nuclei Wash Buffer without Tween-20, nuclei were taken up in 100 µl of 1x PBS containing 1% w/v BSA and 1% v/v SUPERase-In RNase Inhibitor. We used 5 µl for cell counting in duplicates on a CASY device (Schärfe Systems) and diluted to 5,000 nuclei per µl with PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq."
Reverse Transcription: 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and obtained at 100 µM concentration in EB Buffer on 96-well plates (sequences are provided in Supplementary Table 1). 384-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at -20 ˚C (1 µl of 25 µM per well). 10,000 permeabilized cells or nuclei (2 µl of a 5,000/µl suspension) were added to the pre-dispensed primers in each well, and the assignment of samples to wells was recorded. The plate was incubated for 5 min at 55 ˚C to resolve RNA secondary structures, then placed immediately on ice to prevent their re-formation. Per well, a mix of 3 µl nuclease-free water, 2 µl 5x Reverse Transcription Buffer, 0.5 µl of 100 mM DTT (freshly diluted from Sigma #646563-10x.5ML), 0.5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 0.5 µl of RNaseOUT RNase inhibitor (40 U/ml, Thermo Fisher Scientific #10777019), and 0.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753) were added. The reverse transcription was incubated as follows (with heated lid set to 60 ˚C): 50 ˚C for 10 min, 3 cycles of [8 ˚C for 12 s, 15 ˚C for 45 s, 20 ˚C for 45 s, 30 ˚C for 30 s, 42 ˚C for 2 min, 50 ˚C for 3 min], 50 ˚C for 5 min, store at 4 ˚C.  Cell/Nuclei recovery and pooling: Processed cells/nuclei were recovered from the 384-well plate and pooled in one 15 ml tube per plate on ice. Wells were washed with ice-cold 1x PBS containing 1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 15 ml with 1x PBS containing 1% BSA, and nuclei were collected (500 rcf, 5 min, 4 ˚C). The resulting pellet was resuspended in 1.0 ml of 1x Ampligase Reaction Buffer (Lucigen #A0102K), filtered through a cell strainer (40 µm or 70 µm depending on the cell/nuclei size) into a 1.5 ml tube, and centrifuged (500 rcf, 5 min, 4 ˚C). The supernatant was removed almost completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4 ˚C) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in ~10 µl of a highly concentrated suspension, which was diluted 1:200 with 1x Ampligase Buffer and counted in a Fuchs Rosenthal counting chamber (Incyto #DHC-F01). The desired number of cells/nuclei was brought to a volume of 15 µl with 1x Ampligase Reaction Buffer (Lucigen #A0102K).  Microfluidic thermoligation barcoding: Unused channels in the Chromium Chip E (10x Genomics #2000121) were filled with 75 µl (inlet 1), 40 µl (inlet 2), and 240 µl (inlet 3) of 50% glycerol solution (Sigma #G5516-100ML). Right before loading the chip, a mix of 47.4 µl nuclease-free water, 11.5 µl of 10x Ampligase Reaction Buffer (Lucigen #A0102K), 2.3 µl of 100 U/µl Ampligase (Lucigen #A0102K), 1.5 µl of Reducing Agent B (10x Genomics #2000087), and 2.3 µl of 100 µM Bridge Oligo (sequence provided in Supplementary Table 1) was added per sample. The microfluidic chip was loaded with 75 µl of cells or nuclei in thermoligation mix (inlet 1), 40 µl of Single Cell ATAC Gel Beads (inlet 2, 10x Genomics #2000132), and 240 µl of Partitioning Oil (inlet 3, 10x Genomics #220088) and run on the Chromium system. For thermoligation barcoding, the droplet emulsion was incubated as follows (heated lid set to 105 ˚C, volume set to 100 µl): 12 c


title: PD212_scifi_2C_4lines_7650_MeOH-cells

description:
Fig. 2e-h, Fig. S8a-f, Fig. S9, Fig. S10, Fig. S11
Technology comparison: scifi-RNA-seq 7.5k methanol-fixed cells per microfluidic channel


title: PD209_N_scATAC-NextGEM_libprep-std

description:
Fig. S7
scifi-RNA-seq on the scATAC v1.1 (NextGEM) platform using cells or nuclei, and either Superscript IV or Maxima H Minus as reverse transcriptase


title: SE21s_ZT3

description:
mRNA profiling
AllSamples_FPKM.txt


title: PD193_humanmouse_765k

description:
Fig. 2a-d
Human (Jurkat) / Mouse (3T3) mixed 1:1, loaded 765k nuclei per microfluidic channel


title: PD212_scifi_1N_4lines_7650_nuclei

description:
Fig. 2e-h, Fig. S8a-f, Fig. S9, Fig. S10, Fig. S11
Technology comparison: scifi-RNA-seq 7.5k nuclei per microfluidic channel


title: SE21s_ZT0

description:
mRNA profiling
AllSamples_FPKM.txt


title: WT 1

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_wt_vs_5xfad_norm_counts (SL357713)


title: PD190_humanmouse

description:
Fig. 2a-b
Human (Jurkat) / Mouse (3T3) mixed 1:1, loaded 383k nuclei per microfluidic channel, this sample uses only 96 round1 indices


title: PD193_fourlines_383k

description:
Fig. 2i-l, Fig. S12
Large scale experiment on 4 human cell lines (HEK293T, K562, Jurkat, NALM6) mixed 1:1:1:1


title: PD195-1_Tcells_765k-sample1_P7

description:
Fig. S13
Primary human T cells

source_name: Human primary CD3+ T cells

growth_protocol: For each donor, we prepared T cells from 3x 15 ml of peripheral blood, according to the following protocol. 15 ml of peripheral blood were mixed with 750 µl of RosetteSep Human T Cell Enrichment Cocktail (Stemcell #15061). After 10 min of incubation at room temperature, the sample was diluted by addition of 15 ml 1x PBS (Gibco #14190-094) containing 2% v/v FCS (Sigma). SepMate tubes (Stemcell #86450) were loaded with 15 ml of Lymphoprep density gradient medium (Stemcell #07851) and the blood sample was poured on top. After centrifugation (1,200 rcf, 10 min, room temperature, brake set to 9 out of 9), the supernatant was transferred to a fresh 50 ml tube, topped up to 50 ml with 1x PBS containing 2% FCS, and centrifuged (1200 rcf, 10 min, room temperature, brake set to 3 out of 9). After one additional wash with 50 ml of 1x PBS containing 2% FCS (1200 rcf, 10 min, room temperature, brake set to 3), T cells were resuspended in 10 ml of 1x PBS containing 2% FCS, filtered through a 40 µM cell strainer, and counted using a CASY device (Schärfe Systems). For accurate cell counting, it was important to exclude contaminating erythrocytes, given that these cells are lysed during the subsequent nuclei preparation. Freshly isolated primary human T cells were resuspended at a density of 1 million cells per ml in Human T Cell Medium (OpTmizer medium (Thermo Fisher #A1048501) containing 1/38.5 volumes of OpTmizer supplement, 1x GlutaMax (Thermo Fisher #35050061), 1x Penicillin/Streptomycin (Thermo Fisher #15140122), 2% heat-inactivated human AB serum (Fisher Scientific #MT35060CI), 10 ng/ml of recombinant human IL-2 (PeproTech #200-02)).

extract_protocol:
Fixed nuclei suspension: A total of 5 million primary cells were washed with 10 ml of ice-cold 1x PBS (Gibco #14190-094) with centrifugation (300 rcf, 5 min, 4 ˚C). Nuclei were prepared by resuspending cells in 500 µl of ice-cold Nu-clei Preparation Buffer without Digitonin and without Tween-20 (10 mM Tris-HCl pH 7.5 (Sigma #T2944-100ML), 10 mM NaCl (Sigma #S5150-1L), 3 mM MgCl2 (Ambion #AM9530G), 1% w/v BSA (Sigma #A8806-5), 1% v/v SUPERase-In RNase Inhibitor (Thermo Fisher Scientific #AM2696), 0.1% v/v IGEPAL CA-630 (Sigma #I8896-50ML)), followed by 5 min of incubation on ice. Lysis of the plasma membrane was stopped by addition of 5 ml of Nuclei Wash Buffer without Tween-20 (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor). Nuclei were collected by centrifugation (500 rcf, 5 min, 4 ˚C), and fixed in 5 ml of ice-cold 1x PBS containing 1% Formaldehyde (Thermo Fisher Scientific #28908) for 15 min on ice. Fixed nuclei were collected (500 rcf, 5 min, 4 ˚C), the pellet was resuspended in 1.5 ml of ice-cold Nuclei Wash Buffer without Tween-20 and transferred to a 1.5 ml tube. After one more wash with 1.5 ml of ice-cold Nuclei Wash Buffer without Tween-20 (500 rcf, 5 min, 4 ˚C), fixed nuclei were resuspended in 200 µl of Nuclei Wash Buffer without Tween-20, snap-frozen in liquid nitrogen and stored at -80 ˚C.  For scifi-RNA-seq, frozen samples were thawed in a 37 ˚C water bath for exactly 1 min, and immediately placed on ice. Following centrifugation (500 rcf, 5 min, 4 ˚C), fixed nuclei were resuspended in 250 µl of ice-cold Permeabilization Buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 1% w/v BSA, 1% v/v SUPERase-In RNase Inhibitor, 0.01% v/v Digitonin (Promega #G944A), 0.1% v/v Tween-20 (Sigma #P7949-500ML)). After 5 min of incubation on ice, 250 µl of Nuclei Wash Buffer without Tween-20 were added per sample, and nuclei were collected (500 rcf, 5 min, 4 ˚C). After one more wash with 250 µl of Nuclei Wash Buffer without Tween-20, nuclei were taken up in 100 µl of 1x PBS containing 1% w/v BSA and 1% v/v SUPERase-In RNase Inhibitor. We used 5 µl for cell counting in duplicates on a CASY device (Schärfe Systems) and diluted to 5,000 nuclei per µl with PBS-BSA-SUPERase. We immediately proceeded with the reverse transcription step of scifi-RNA-seq.
Reverse Transcription: 384 indexed reverse transcription primers were synthesized by Sigma Aldrich and obtained at 100 µM concentration in EB Buffer on 96-well plates (sequences are provided in Supplementary Table 1). 384-well plates with barcoded oligo-dT primers were prepared prior to the experiment and stored at -20 ˚C (1 µl of 25 µM per well). 10,000 permeabilized cells or nuclei (2 µl of a 5,000/µl suspension) were added to the pre-dispensed primers in each well, and the assignment of samples to wells was recorded. The plate was incubated for 5 min at 55 ˚C to resolve RNA secondary structures, then placed immediately on ice to prevent their re-formation. Per well, a mix of 3 µl nuclease-free water, 2 µl 5x Reverse Transcription Buffer, 0.5 µl of 100 mM DTT (freshly diluted from Sigma #646563-10x.5ML), 0.5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 0.5 µl of RNaseOUT RNase inhibitor (40 U/ml, Thermo Fisher Scientific #10777019), and 0.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753) were added. The reverse transcription was incubated as follows (with heated lid set to 60 ˚C): 50 ˚C for 10 min, 3 cycles of [8 ˚C for 12 s, 15 ˚C for 45 s, 20 ˚C for 45 s, 30 ˚C for 30 s, 42 ˚C for 2 min, 50 ˚C for 3 min], 50 ˚C for 5 min, store at 4 ˚C.  Cell/Nuclei recovery and pooling: Processed cells/nuclei were recovered from the 384-well plate and pooled in one 15 ml tube per plate on ice. Wells were washed with ice-cold 1x PBS containing 1% BSA, which was transferred to the same tube for maximum recovery. The volume was topped up to 15 ml with 1x PBS containing 1% BSA, and nuclei were collected (500 rcf, 5 min, 4 ˚C). The resulting pellet was resuspended in 1.0 ml of 1x Ampligase Reaction Buffer (Lucigen #A0102K), filtered through a cell strainer (40 µm or 70 µm depending on the cell/nuclei size) into a 1.5 ml tube, and centrifuged (500 rcf, 5 min, 4 ˚C). The supernatant was removed almost completely, and the tube was centrifuged briefly (500 rcf, 30 s, 4 ˚C) to collect the remaining liquid at the bottom of the tube. Typically, this resulted in ~10 µl of a highly concentrated suspension, which was diluted 1:200 with 1x Ampligase Buffer and counted in a Fuchs Rosenthal counting chamber (Incyto #DHC-F01). The desired number of cells/nuclei was brought to a volume of 15 µl with 1x Ampligase Reaction Buffer (Lucigen #A0102K).  Microfluidic thermoligation barcoding: Unused channels in the Chromium Chip E (10x Genomics #2000121) were filled with 75 µl (inlet 1), 40 µl (inlet 2), and 240 µl (inlet 3) of 50% glycerol solution (Sigma #G5516-100ML). Right before loading the chip, a mix of 47.4 µl nuclease-free water, 11.5 µl of 10x Ampligase Reaction Buffer (Lucigen #A0102K), 2.3 µl of 100 U/µl Ampligase (Lucigen #A0102K), 1.5 µl of Reducing Agent B (10x Genomics #2000087), and 2.3 µl of 100 µM Bridge Oligo (sequence provided in Supplementary Table 1) was added per sample. The microfluidic chip was loaded with 75 µl of cells or nuclei in thermoligation mix (inlet 1), 40 µl of Single Cell ATAC Gel Beads (inlet 2, 10x Genomics #2000132), and 240 µl of Partitioning Oil (inlet 3, 10x Genomics #220088) and run on the Chromium system. For thermoligation barcoding, the droplet emulsion was incubated as follows (heated lid set to 105 ˚C, volume set to 100 µl): 12 cycles of [98 ˚C for 30 s, 59 ˚C for 2 min], storage at 15 ˚C. The emulsion was broken by addition of 125 µl Recovery Agent (10x Genomics #220016), and 125 µl of the pink oil phase were removed by pipetting. The remaining sample was mixed with 200 µl of Dynabead Cleanup Master Mix (per reaction: 182 µl Cleanup Buffer (10x Genomics #2000088), 8 µl Dynabeads MyOne Silane (Thermo Fisher Scientific #37002D), 5 µl Reducing Agent B (10x Genomics #2000087), 5 µl of nuclease-free water). After 10 min of incubation at room temperature, samples were washed twice with 200 µl of freshly prepared 80% ethanol (Merck #603-002-00-5) and eluted in 40.5 µl of EB Buffer (Qiagen #19086) containing 0.1% Tween (Sigma #P7949-500ML) and 1% v/v Reducing Agent B. Bead clumps were sheared with a 10 µl pipette or needle. 40 µl of the sample were transferred to a fresh tube strip and subjected to a 1.0x cleanup with SPRIselect beads (Beckman Coulter #B23318), eluting in 22 µl of EB Buffer.  Template switching: 20 µl of sample from the previous step were mixed with 10 µl of 5x Reverse Transcription Buffer, 10 µl of Ficoll PM-400 (20%, Sigma #F5415-50ML), 5 µl of 10 mM dNTPs (Thermo Fisher Scientific #R0193), 1.25 µl of Recombinant Ribonuclease Inhibitor (Takara #2313A), 1.25 µl of 100 µM Template Switching Oligo (sequence provided in Supplementary Table 1), and 2.5 µl of Maxima H Minus Reverse Transcriptase (200 U/ml, Thermo Fisher Scientific #EP0753). The template switching reaction was incubated for 30 min at 25 ˚C, 90 min at 42 ˚C, storage at 4 ˚C, and cleaned with a 1.0x SPRI cleanup, eluting in 17 µl of EB buffer.  cDNA enrichment: 15 µl of the above sample were mixed with 33 µl of nuclease-free water, 50 µl of NEBNext High-Fidelity 2x PCR Master Mix (NEB #M0541S), 0.5 µl of 100 µM Partial P5 primer, 0.5 µl of 100 µM TSO Enrichment Primer (sequences provided in Supplementary Table 1), and 1 µl of 100x SYBR Green in DMSO (Life Technologies #S7563). cDNA was amplified in a thermocycler as follows: 98 ˚C for 30 s, cycle until fluorescent signal >1000 RFU [98 ˚C for 20 s, 65 ˚C for 30 s, 72 ˚C for 3 min], 72 ˚C for 5 min in another thermocycler, storage at 4 ˚C. cDNA was cleaned by one 0.8x SPRI cleanup followe

treatment_protocol: The culture was split into two flasks, and one was treated with Human T-Activator CD3/CD28 Dynabeads (25 µl beads per 1 million cells, Thermo Fisher #11131D). After 16h we proceeded with the scifi-RNA-seq library preparation.


title: WT 2

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_wt_vs_5xfad_norm_counts (SL357714)


title: pLKO.1 Scramble (replicate#1)

status: Public on Mar 10 2021

type: RNA

city: Verona

first: Mirco

last: Galiè

address: via Le Grazie, 8

department: Neuroscience, Biomedicine and Movement Sciences

country: Italy

institute: University of Verona

hyb_protocol: standard Affymetrix protocol

scan_protocol: standard Affymetrix protocol

data_processing: RMA normalization using Expression Console 1.4.1.46

label: biotin

source_name: pLKO.1 Scramble_MSCs

label_protocol: standard Affymetrix protocol

growth_protocol: MSC maintained in DMEM (high-glucose), GLUTAMAX I-10%FBS-100U/ml Penicillin and 100ug/ml Streptomycin

extract_protocol: RNA extracted using Qiagen Rneasy kit, closely following manifacturer's instructions.

value: FVB

tag: gender

value: 12 months

value: inguinal fat pad

tag: cell type

value: mesenchymal stromal cells (MSCs)

tag: treatment/genotype

value: pLKO.1 Scramble


title: pLKO.1 shIRF7 (replicate#1)

source_name: pLKO.1 shIRF7_MSCs

value: pLKO.1 shIRF7


title: pLKO.1 shIRF7 (replicate#4)


title: Samp11-Preexposure-Myeloma

city: Northfield

first: Heather

middle: Elizabeth

last: Driscoll

state: VT

address: 158 Harmon Drive

department: Biology

institute: Norwich University

description: Second of three experimental replicates of differentiated 3T3-L1 adipocyte cells in culture exposed to myeloma MM1S cells prior to differentiation.

hyb_protocol: Samples were combined with a hybridization mix, injected into Mouse ClariomTM S arrays, and placed in the Affymetrix® GeneChip® Hybridization Oven 645 at 45° C and 60 RPM for 16.5 hours overnight.

scan_protocol: Arrays were stained using the Affymetrix® GeneChip® Fluidics Station 450 and scanned with the 7G Affymetrix® GeneChip® Scanner 3000. Raw image analysis was performed with Affymetrix® Expression ConsoleTM software. The raw data images produced from the scanner were processed into .CEL files, which contain measure intensities for each probe on the array.

data_processing: The .CEL files were imported into Partek® Genomics SuiteTM (GS) version 6.6 (Partek Inc., St. Louis, MO, USA, www.partek.com). Background correction, quantile normalization, log2 transformation, and probe set summarization were performed using default settings for the Robust Multichip Average (RMA) procedure (see Bolstad et al. Bioinformatics 19(2):185; Irizarry et al. Biostatistics 4(2):249; Nucleic Acids Res. 31:e15). A two-way Analysis of Variance (ANOVA) with factors matrix type and subject (paired design) was performed on the RMA-normalized probesets to assess differential expression.

label: Biotin

source_name: Adipose 3T3-L1 cell culture after 7 days of adipogenic differentiation with pre-exposure to myeloma MM1S cells

label_protocol: Approximately 5.5µg of single-stranded cDNA was fragmented using UDG (10U/µL) and APE1 (1000U/µL), provided in the GeneChip® WT PLUS Reagent Kit. Samples were then labeled with biotin using TdT (30U/µL), also provided in the GeneChip® WT PLUS Reagent Kit. Efficiency of the fragmentation and labeling reactions were verified using NeutrAvidin (10mg/mL) with a gel-shift assay.

growth_protocol: Preadipocytes were cultured in basal growth media (DMEM supplemented with 10% FBS and 1% penicillin/streptomycin) for 4 days and then switched to an adipogenic differentiation media (basal supplemented with 0.5 mM IBMX, 1 µM Dex, 5 µg/mL insulin) for 4 days, and then a second adipogenic media (basal supplemented with 5 µg/mL insulin) for 2 days and then maintained in basal media for an additional day to ensure terminal differentiation into adipocytes.

extract_protocol: After differentiation and myeloma exposure periods, total RNA was isolated from 10 samples using the Qiazol and miRNeasy isolation mini-kit (Qiagen) according to the manufacturer’s protocol. 50ng of RNA was used to synthesize cDNA through a first-strand reverse transcription reaction and second-strand RNA degradation reaction, using reagents from the GeneChip® WT PLUS Reagent Kit. cRNA was then synthesized through an overnight (16-hour) in-vitro transcription reaction, which utilizes a T7 RNA polymerase. The cRNA was purified using an Affymetrix® magnetic bead protocol. Sample concentrations were determined using a 40µg/mL/A260 constant on a Nanodrop 1000 spectrophotometer.

treatment_protocol: For the pre-exposure experiment, after two days in basal growth media pre-adipocytes reached confluency and were switched to RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin either alone, or with MM.1S myeloma cells for 48 hours. At this time, myeloma cells were removed, pre-adipocytes were washed, and then differentiated as outlined above. For the mature adipocyte experiment, 3T3-L1 pre-adipocytes were cultured exactly as outlined above. After differentiation, adipocytes were changed to RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin either alone, or with MM.1S myeloma cells for 72 hours. In both experiments myeloma cells were co-cultured with 3T3-L1 cells indirectly utilizing a 0.4 µm transwell membrane.

tag: experiment

value: Experiment 1

value: Pre-adipocyte Cell Line

tag: adipocyte developmental stage

value: Mature

tag: exposure to mm1s myeloma cell line

value: Pre-exposure


title: pLKO.1 shIRF7 (replicate#3)


title: pLKO.1 Scramble (replicate#2)


title: Don3.CD8_N.IL27

city: Montréal

first: Nathalie

last: Arbour

state: Quebec

address: 900 Saint Denis Street

department: Neuroscience

country: Canada

institute: CHUM-University of Montreal

hyb_protocol: After fragmentation and labeling, 2.8 µg DNA target was hybridized on Clariom™S HT, Human (Thermo Fisher Scientific)

scan_protocol: Arrays were scanned on the GeneTitan® Instrument (Thermo Fisher Scientific) for Hyb-Wash-Scan automated workflow.

data_processing: Data was analyzed using R. Normalization of CEL files was done using the function rma from the oligo Bioconductor package. Differential expression analysis was performed using the limma Bioconductor package with linear modeling, accounting for thee paired nature of samples. Significance was assessed  as followed: absolute fold-change > 1.3 and adjusted p-value < 0.15

source_name: PBMC, isolated naive CD8 T cells

label_protocol: Sense-strand cDNA was synthesized from 100 ng of total RNA, and fragmentation and labeling were performed to produce ss-cDNA with the GeneChip® WT Terminal Labeling Kit according to manufacturer’s instructions (Thermo Fisher Scientific).

growth_protocol: Naive (CD45RA+CD45RO-CCR7+) and central memory (CD45RA-CD45RO+CCR7+) CD4+ and CD8+ T cells from 3 healthy donors were first sorted on a BD FACS Aria III and put in culture in Iscove's Modified Dulbecco's Medium supplemented with 10% (v/v) FBS, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 ug/mL), sodium pyruvate (1mM), non-essential amino acids (1X solution), and β-mercaptoethanol (50µM).

extract_protocol: RNA samples were purified using RNeasy plus mini kit according to the manufacturer’s protocol (Qiagen, Toronto, ON Canada). RNA quality was checked on RNA Nano chips using a 2100 bioanalyser (Agilent Technologies). All samples had a RNA integrity number (RINs) > 8.3.

treatment_protocol: Sorted naive and central memory CD4+ and CD8+ T cells were stimulated for 3 days with immobilized αCD3 (Naive: 1.6 µg/mL, Central memory: 0.32 µg/mL) and soluble αCD28 (1 µg/mL) in the presence or not of IL-27 (100 ng/mL).

value: M

tag: stimulation

value: with IL-27

value: PBMC, isolated naive CD8 T cells


title: Don3.CD4_CM.NIL

source_name: PBMC, isolated central memory CD4 T cells

value: without IL-27


title: Don3.CD8_CM.NIL

source_name: PBMC, isolated central memory CD8 T cells


title: pLKO.1 Empty vector (replicate#2)

source_name: pLKO.1 Empty vector_MSCs

tag: strain

value: male

value: pLKO.1 Empty vector


title: pLKO.1 shIRF7 (replicate#2)

tag: tissue


title: pLKO.1 Empty vector (replicate#1)


title: Samp10-Preexposure-Control

description: Second of two control replicates of 3T3-L1 adipocyte cells in culture.

source_name: Adipose 3T3-L1 cell culture after 7 days of adipogenic differentiation and no exposure to myeloma MM1S cells

tag: cell line


title: Samp4-Preexposure-Myeloma

description: First of three experimental replicates of differentiated 3T3-L1 adipocyte cells in culture exposed to myeloma MM1S cells prior to differentiation.


title: Don3.CD8_CM.IL27

value: PBMC, isolated central memory CD8 T cells


title: MCC13 [methylation]

type: genomic

city: Essen

first: Jan

last: Gravemeyer

address: Universitätstraße 1

country: Germany

institute: DKZF

description: DNA methylation measured by the Infinium EPIC BeachChip

hyb_protocol: The infinium EPIC BeachChip assay was performed at the microarray unit of the DKFZ Genomics and Proteomics Core Facility with > 1000ng of input DNA and following  the standard illumina protocol

data_processing: Raw IDAT files were processed in R following the cross-package workflow described by Maksimovic et al. This included the Bioconductor packages minfi  and limma . Quality of the data was assessed with the minfi qcReport function. Data were normalized using functional normalization  implemented in minfi. Probes with a detection p-value above 0.01 in at least one sample were removed. Additionally, probes located on sex chromosomes, probes with SNPs at CpG sites and cross-reactive probes were discarded

label: Cy5 and Cy3

molecule: genomic DNA

source_name: cell line

growth_protocol: The vMCC (MCC13, MCC26, UISO ) and cMCC (WaGa, PeTa, MKL-1, MKL-2, and UKE-MCC-1a) cell lines were maintained in RPMI-1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (Sigma, St. Lous, MO, USA) and 1% penicillin/streptomycin (Pan Biotech, Aidenbach, Germany). The SCC cell lines (Met-1, HSC-1, SCL-1) were maintained in DMEM (Pan Biotech, Aidenbach, Germany), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

extract_protocol: DNA isolation from cell lines was performed using Qiagen QiAamp DNA mini Kit according to the manufacturer’s protocol

treatment_protocol: no treatment

tag: sample type

value: variant Merkel cell carcinoma


title: UISO [methylation]

scan_protocol: The infinium EPIC BeachChip assay was performed at the microarray unit of the DKFZ Genomics and Proteomics Core Facility with > 1000ng of input DNA and following  the standard illumina protocol


title: PeTa [methylation]

label_protocol: The infinium EPIC BeachChip assay was performed at the microarray unit of the DKFZ Genomics and Proteomics Core Facility with > 1000ng of input DNA and following  the standard illumina protocol

value: classical Merkel cell carcinoma


title: Samp1-Preexposure-Control

description: First of two control replicates of differentiated 3T3-L1 adipocyte cells in culture.


title: Don3.CD4_N.IL27

source_name: PBMC, isolated naive CD4 T cells

value: PBMC, isolated naive CD4 T cells


title: Don3.CD4_CM.IL27

value: PBMC, isolated central memory CD4 T cells


title: MKL-2 [methylation]


title: Don3.CD8_N.NIL


title: HSC-1 [methylation]

value: Squamous cell carcinoma


title: SCL-1 [methylation]


title: Don2.CD4_CM.NIL

value: F


title: Don2.CD8_N.IL27


title: Don2.CD8_N.NIL


title: Don2.CD8_CM.IL27


title: WaGa [methylation]


title: MKL-1 [methylation]


title: Don2.CD4_N.IL27


title: Don1.CD8_CM.IL27


title: Don2.CD4_N.NIL


title: Don2.CD8_CM.NIL


title: Don1.CD8_CM.NIL


title: UKE-MCC-1a [methylation]


title: Don1.CD8_N.IL27


title: Don3.CD4_N.NIL


title: Met-1 [methylation]


title: MCC26 [methylation]


title: Don1.CD8_N.NIL


title: Hypothalamus Negative Control

city: Bloomington

first: Kimberly

last: Rosvall

state: IN

address: 1001 E Third Street

institute: Indiana University

description: Negative Control

library_source: genomic

data_processing:
Demultiplexing of the reads was performed with bcl2fastq version 2.20.0 pipeline
Adapters and low quality bases were trimmed using Trimmomatic (version 0.36) with following specific parameters ILLUMINACLIP:<adapters.fa>:2:20:5 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:17
RNA-Seq reads were mapped to the reference tree swallow transcriptome obtained previously by our laboratory (Bentz et al. 2019) using Bowtie2 (version 2.3.4.3)
Reads mapped to transcripts were counted using Samtools (version 1.9 )
For RNA-seq, R (version 3.6.0) and DESeq2 were used to perform differential expresison analysis.
MeCap reads were mapped to the reference tree swallow genome, GCA_007636935.1, using bwa (version 0.7.17-r1188). Duplicate reads were marked and removed using Picard Tools MarkDuplicates (version 2.20.3-SNAPSHOT) and Samtools version (1.9)
Inserts were inferred from the concordantly mapped, deduplicated read alignments. Number of inserts spanning over each base position were estimated and averaged over 250bp fixed length non-overlapping, contiguous bins. The average bin coverage values for each library were normalized based on the total number of inserts inferred in that library.
tree swallow transcriptome obtained previously by our laboratory (Bentz et al. 2019)
Genome_build: Tree Swallow genome GCA_007636935.1,  https://www.ncbi.nlm.nih.gov/assembly/GCA_007636935.1
Supplementary_files_format_and_content: rnaseq_final_transcripts_counts.tsv is a tab-delimited text file of raw RNA-seq read count values for each Sample
Supplementary_files_format_and_content: mecap_normalized_coverage_per_bin.tsv contains the normalized coverage of MeCap reads mapped to the tree swallow genome in 250bp windows

organism: Tachycineta bicolor

source_name: Hypothalamus

extract_protocol:
Brains were frozen on dry ice in the field and macrodissected into functional regions. Total RNA was extracted from each sample using Trizol (Invitrogen, Carlsbad, CA). Genomic DNA was isolated from each sample using a Quick-DNA Microprep Plus Kit (Zymo Research) and 300ng of DNA was sheared to an average size of 200bp using a Covaris E220 sonicator (Covaris). DNA Methyl capture was performed using a MethylCap kit (Diagenode). A negative control (the methyl binding domain protein was excluded during Methyl capture) and a fully methylated positive control (DNA treated with SssI CpG methyltransferase; New England Biolabs) were included.
Total RNA was used for cDNA library construction using a TruSeq Stranded mRNA kit (Illumina) following the kit protocol. MethylCap-Seq libraries were generated using the NextFlex MethylSeq Kit (Bioo Scientific) following the kit protocol
RNA-Seq and MethylCap-Seq

treatment_protocol: Experimental: temporarily removed nest boxes to increase competition among free-living birds; Control: date-matched unmanipulated birds

value: brain

tag: developmental stage

value: adult

value: female


title: Don1.CD4_CM.IL27


title: Don1.CD4_N.NIL


title: Don1.CD4_N.IL27


title: Don2.CD4_CM.IL27


title: SAMPLE 6: Homo sapiens Esophageal squamous cell carcinoma cells KYSE30 siRNA against MTA2

city: Shijiazhuang,Hebei,China

first: Suli

last: Dai

address: No. 12 Jiankang Road, Shijiazhuang, Hebei

institute: The Fourth Hospital of Hebei Medical University

description: Homo sapiens esophageal squamous cell carcinoma cells KYSE30 transfected with siRNA against MTA2 (MTA2-homo-409:Sense：5'-3' GGUGGGAGAUUACGUCUAUTT, Antisense：5'-3' AUAGACGUAAUCUCCCACCTT)

hyb_protocol: Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.

data_processing: Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis.

label_protocol: Total RNA of each sample was used for labeling and array hybridization as the following steps: 1) Reverse transcription with by Invitrogen Superscript ds-cDNA synthesis kit; 2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit; 3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the NimbleGen wash buffer kit; 4) Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation).

growth_protocol: Homo sapiens Esophageal Squamous Cell carcinoma cells KYSE30 was grown to Logarithmic phase phase (5% CO2,at 37°C ) in RPMI1640 (Gibco, Eggenstein, Germany) supplemented with 10% (vol) fetal bovine serum (10106078, Gibco) and 100U/ml penicillin

extract_protocol: Total RNA was extracted using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) and the RNAprotect reagent (Qiagen) and DNA was removed by on-column DNase digestion with the RNase-Free DNase set (Qiagen). Total RNA from each sample was quantified by the NanoDrop ND-1000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis.

treatment_protocol: Homo sapiens Esophageal Squamous Cell carcinoma cells KYSE30 was transfected with siRNA negative control or siRNA against  MTA2 20nmol/ml for 48h using Lipofectamine 2000.


title: Hypothalamus DNA_INTSF37_EXP_Day 2

description: DNA_INTSF37_EXP_Day 2

value: Experimental

tag: period

value: Day 2


title: Hypothalamus DNA_INTSF4_EXP_Day 0

description: DNA_INTSF4_EXP_Day 0

value: Day 0


title: Hypothalamus DNA_INTSF5_EXP_Day 0

description: DNA_INTSF5_EXP_Day 0


title: Hypothalamus DNA_INTSF24_CON_Day 2

description: DNA_INTSF24_CON_Day 2

value: Control


title: Samp20-Preexposure-Myeloma

description: Third of three experimental replicates of differentiated 3T3-L1 adipocyte cells in culture exposed to myeloma MM1S cells prior to differentiation.

value: Experiment 1

value: Pre-adipocyte Cell Line

value: Mature

value: Pre-exposure


title: Hypothalamus DNA_INTSF52_EXP_Day 2

description: DNA_INTSF52_EXP_Day 2


title: Hypothalamus DNA_INTSF18_EXP_Day 0

description: DNA_INTSF18_EXP_Day 0


title: Hypothalamus DNA_INTSF50_EXP_Day 2

description: DNA_INTSF50_EXP_Day 2


title: Hypothalamus DNA_INTSF15_EXP_Day 0

description: DNA_INTSF15_EXP_Day 0


title: Hypothalamus DNA_INTSF22_CON_Day 2

description: DNA_INTSF22_CON_Day 2


title: Hypothalamus Positive Control

description: Positive Control


title: Hypothalamus DNA_INTSF12_EXP_Day 2

description: DNA_INTSF12_EXP_Day 2


title: SAMPLE 3: Homo sapiens Esophageal squamous cell carcinoma cells KYSE30 siRNA control

description: Homo sapiens esophageal squamous cell  carcinoma celsl KYSE30 transfected with siRNA normal control (Sense：5'-3'UUCUCCGAACGUGUCACGUTT, Antisense：5'-3'ACGUGACACGUUCGGAGAATT). It is the control group

scan_protocol: Array scanning using the Axon GenePix 4000B microarray scanner (Molecular Devices Corporation).


title: Hypothalamus DNA_INTSF16_EXP_Day 0

description: DNA_INTSF16_EXP_Day 0


title: Hypothalamus DNA_INTSF3_EXP_Day 0

description: DNA_INTSF3_EXP_Day 0


title: Hypothalamus DNA_INTSF38_CON_Day 2

description: DNA_INTSF38_CON_Day 2


title: Hypothalamus DNA_INTSF23_CON_Day 2

description: DNA_INTSF23_CON_Day 2


title: SAMPLE 5: Homo sapiens Esophageal squamous cell carcinoma cells KYSE30 siRNA against MTA2


title: Ventromedial Telencephalon RNA_INTSF50_EXP_Day 2

description: RNA_INTSF50_EXP_Day 2

source_name: Ventromedial Telencephalon


title: Hypothalamus DNA_INTSF6_CON_Day 0

description: DNA_INTSF6_CON_Day 0


title: Hypothalamus DNA_INTSF11_CON_Day 0

description: DNA_INTSF11_CON_Day 0


title: SAMPLE 4: Homo sapiens Esophageal squamous cell carcinoma cells KYSE30 siRNA against MTA2


title: Ventromedial Telencephalon RNA_INTSF52_EXP_Day 2

description: RNA_INTSF52_EXP_Day 2


title: Hypothalamus DNA_INTSF19_CON_Day 2

description: DNA_INTSF19_CON_Day 2


title: Ventromedial Telencephalon RNA_INTSF12_EXP_Day 2

description: RNA_INTSF12_EXP_Day 2


title: SAMPLE 2: Homo sapiens Esophageal squamous cell carcinoma cells KYSE30 siRNA control


title: SAMPLE 1: Homo sapiens Esophageal squamous cell carcinoma cells KYSE30 siRNA control

description: Homo sapiens esophageal squamous cell  carcinoma cells KYSE30 transfected with siRNA normal control (Sense：5'-3'UUCUCCGAACGUGUCACGUTT, Antisense：5'-3'ACGUGACACGUUCGGAGAATT). It is the control group


title: Ventromedial Telencephalon RNA_INTSF4_EXP_Day 0

description: RNA_INTSF4_EXP_Day 0


title: Hypothalamus DNA_INTSF8_CON_Day 0

description: DNA_INTSF8_CON_Day 0


title: Hypothalamus DNA_INTSF9_CON_Day 0

description: DNA_INTSF9_CON_Day 0


title: Hypothalamus DNA_INTSF7_CON_Day 0

description: DNA_INTSF7_CON_Day 0


title: Ventromedial Telencephalon RNA_INTSF37_EXP_Day 2

description: RNA_INTSF37_EXP_Day 2


title: Ventromedial Telencephalon RNA_INTSF18_EXP_Day 0

description: RNA_INTSF18_EXP_Day 0


title: Ventromedial Telencephalon RNA_INTSF16_EXP_Day 0

description: RNA_INTSF16_EXP_Day 0


title: Ventromedial Telencephalon RNA_INTSF38_CON_Day 2

description: RNA_INTSF38_CON_Day 2


title: Ventromedial Telencephalon RNA_INTSF24_CON_Day 2

description: RNA_INTSF24_CON_Day 2


title: Ventromedial Telencephalon RNA_INTSF19_CON_Day 2

description: RNA_INTSF19_CON_Day 2


title: Ventromedial Telencephalon RNA_INTSF8_CON_Day 0

description: RNA_INTSF8_CON_Day 0


title: Ventromedial Telencephalon RNA_INTSF9_CON_Day 0

description: RNA_INTSF9_CON_Day 0


title: Ventromedial Telencephalon RNA_INTSF7_CON_Day 0

description: RNA_INTSF7_CON_Day 0


title: Ventromedial Telencephalon RNA_INTSF3_EXP_Day 0

description: RNA_INTSF3_EXP_Day 0


title: Ventromedial Telencephalon RNA_INTSF5_EXP_Day 0

description: RNA_INTSF5_EXP_Day 0


title: Hypothalamus RNA_INTSF37_EXP_Day 2

description: RNA_INTSF37_EXP_Day 2


title: Ventromedial Telencephalon RNA_INTSF22_CON_Day 2

description: RNA_INTSF22_CON_Day 2


title: Hypothalamus RNA_INTSF50_EXP_Day 2

description: RNA_INTSF50_EXP_Day 2


title: Ventromedial Telencephalon RNA_INTSF11_CON_Day 0

description: RNA_INTSF11_CON_Day 0


title: Hypothalamus RNA_INTSF12_EXP_Day 2

description: RNA_INTSF12_EXP_Day 2


title: Hypothalamus RNA_INTSF3_EXP_Day 0

description: RNA_INTSF3_EXP_Day 0


title: Hypothalamus RNA_INTSF52_EXP_Day 2

description: RNA_INTSF52_EXP_Day 2


title: Hypothalamus RNA_INTSF4_EXP_Day 0

description: RNA_INTSF4_EXP_Day 0


title: Hypothalamus RNA_INTSF18_EXP_Day 0

description: RNA_INTSF18_EXP_Day 0


title: Hypothalamus RNA_INTSF16_EXP_Day 0

description: RNA_INTSF16_EXP_Day 0


title: Hypothalamus RNA_INTSF15_EXP_Day 0

description: RNA_INTSF15_EXP_Day 0


title: Ventromedial Telencephalon RNA_INTSF6_CON_Day 0

description: RNA_INTSF6_CON_Day 0


title: Hypothalamus RNA_INTSF38_CON_Day 2

description: RNA_INTSF38_CON_Day 2


title: Ventromedial Telencephalon RNA_INTSF23_CON_Day 2

description: RNA_INTSF23_CON_Day 2


title: Hypothalamus RNA_INTSF5_EXP_Day 0

description: RNA_INTSF5_EXP_Day 0


title: Hypothalamus RNA_INTSF23_CON_Day 2

description: RNA_INTSF23_CON_Day 2


title: Hypothalamus RNA_INTSF22_CON_Day 2

description: RNA_INTSF22_CON_Day 2


title: Ventromedial Telencephalon RNA_INTSF15_EXP_Day 0

description: RNA_INTSF15_EXP_Day 0


title: Hypothalamus RNA_INTSF8_CON_Day 0

description: RNA_INTSF8_CON_Day 0


title: Hypothalamus RNA_INTSF19_CON_Day 2

description: RNA_INTSF19_CON_Day 2


title: Hypothalamus RNA_INTSF9_CON_Day 0

description: RNA_INTSF9_CON_Day 0


title: Hypothalamus RNA_INTSF24_CON_Day 2

description: RNA_INTSF24_CON_Day 2


title: Hypothalamus RNA_INTSF6_CON_Day 0

description: RNA_INTSF6_CON_Day 0


title: Hypothalamus RNA_INTSF11_CON_Day 0

description: RNA_INTSF11_CON_Day 0


title: Hypothalamus RNA_INTSF7_CON_Day 0

description: RNA_INTSF7_CON_Day 0


title: Don1.CD4_CM.NIL

tag: age

value: without IL-27


title: cARKO_DHT_rep1

city: Toon

first: Yuuki

last: Imai

state: Ehime

address: Shitsukawa

department: Proteo-Science Center

country: Japan

institute: Ehime University

data_processing:
Sequence data were mapped on the mouse genome (mm10) using Tophat and analyzed by featureCounts.
Genome_build: mm10

source_name: gastrocunemius muscle

growth_protocol: Mice were generated using AR flox mice and HSA-Cre mice and littermate were conducted to the treatment.

extract_protocol:
Total RNA was extracted with Isogen (Nippon gene, Japan) and RNeasy spin column kit (Qiagen, USA). 1 µg of RNA samples with RNA integrity number (RIN) greater than 8 were used for library prepairation.
RNA-Seq libraries were prepared using the Illumina TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego CA) according to the manufacturer's instructions and subsequently validated for an average size of about 305-310 bp using a 2100 Bioanalyzer and the Agilent DNA1000 kit for the construction of sequencing libraries.

treatment_protocol: Nine-week-old female mice were anesthesized and sham-operated or implanted DHT pellet for 4 weeks.

value: 13 weeks old

value: implanted DHT


title: cARKO_Sham_rep3

value: sham-operated


title: cARKO_DHT_rep3


title: Control_DHT_rep3


title: Control2

city: Newark

first: Deepti

last: Anand

state: Delaware

address: 351 Wolf Hall, University of Delaware

department: Biological Sciences

institute: University of Delaware

hyb_protocol: Standard affymetrix protocol

scan_protocol: Standard affymetrix protocol

data_processing: The data were normalised using RMA method  in R

source_name: Lens tissue

label_protocol: Standard protocol for affymetrix arrays

extract_protocol: RNA isolation protocol by mirVanaTM RNA isolation kit (Life Technologies, Grand Island, NY)

value: Post-natal (P4)

tag: genotype

value: wildtype


title: cARKO_Sham_rep1

tag: treatment


title: Control3


title: KO3


title: Control1


title: Control_DHT_rep1


title: Control_DHT_rep2


title: cARKO_Sham_rep2


title: Control_Sham_rep3


title: Control_Sham_rep2


title: KO2


title: KO1


title: Heart circRNA-seq NF2

city: shanghai

first: Zhenhao

last: Lin

address: shanghaixuhui

institute: Fudan University

data_processing:
Library strategy: circRNA-seq
Basecalls performed using CASAVA version 1.8
circRNA-seq reads were mapped to human genome by BWA-MEM software by default parameters except -T = 19
circRNAs were detected and annotated with CIRI software
Genome_build: hg19
Supplementary_files_format_and_content: Number of raw junction read

source_name: Heart

extract_protocol: Construction of the circRNA libraries: Briefly, total RNA was used for removing the rRNAs with NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer's instructions. RNA libraries were constructed by using rRNA-depleted RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) according to the manufacturer's instructions. Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, USA).

value: heart

tag: Sex

value: male

tag: diagnosis

value: healthy


title: Control_Sham_rep1

tag: gender

value: female


title: Heart circRNA-seq NF3


title: HX-3

city: Cincinnati

first: Zaili

last: Luo

state: OH

address: 3333 Burnet Ave.

institute: CCHMC

data_processing:
The fastq data files from RNA-seq were processed by eXpress, a software package for efficient probabilistic assignment of ambiguously mapping sequenced fragments (Roberts and Pachter, 2013) with reference gene annotation from Gencode v29 (Harrow et al., 2012).
The datasets were imported into DESeq2 (Anders and Huber, 2010) to get normalized expression matrix and differentiated expression statistics. We followed the pipeline from Arriba (https://github.com/suhrig/arriba/) for the detection of gene fusions from RNA-Seq data.
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

source_name: primary tissue

extract_protocol:
Medulloblastoma tumor or para-tumor tissues were flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Tumor

tag: medulloblastoma subgroup


title: HX5

value: SHH


title: HX-2

description:
Sample_-2_tissue
2_S1


title: zho19002

city: Cleveland

first: Ernest

middle: R

last: Chan

address: 10900 Euclid Ave, Robbins E211

department: PQHS

institute: CWRU

description: Gene expression data from off-fucose mouse spleen cDC1

hyb_protocol: 5.5 ug of cDNAs were hybridized to Affymetrix Gene Chip Mouse Gene 2.0 ST Array

scan_protocol: GeneChips were scanned using Affymetrix GeneChip Scanner 3000.

data_processing: Normalized robust multi-array average (RMA) values were calculated and used to calculate fold change for each gene and sorted by log2 fold change. A total of 327 genes were identified with a fold change greater than 2 (log2FC > 1) between cDC1 enriched cells isolated from on-fucose and off-fucose mice.  The 327 genes were cross referenced against the Molecular Signature Database (MSigDB, Broad Institute) to identify gene sets enriched in differentially expressed genes.

source_name: Off-fucose mouse spleen cDC1 cells

label_protocol: RNA was reverse transcribed and labeled according to the instruction of Affymetrix WT Pico protocol by using 9 ng total RNA (GeneChip WT Pico Reagent Kit, Thermo Fisher Scientific, Inc)

growth_protocol: cDC1s are freshly isolated from mouse spleens

extract_protocol: Total RNA was extracted from spleen cDC1 cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

treatment_protocol: The total RNA from cDC1 cells were directly extracted

value: spleen

tag: cell type

value: no fucose treatment


title: Heart circRNA-seq NF1


title: Heart circRNA-seq F3

value: dilated cardiomyopathy


title: cARKO_DHT_rep2


title: Heart circRNA-seq F2


title: zho19001

description: Gene expression data from on-fucose mouse spleen cDC1

source_name: On-fucose mouse spleen cDC1 cells

value: fucose-treated


title: Heart circRNA-seq F1


title: K3: modified K562-expanded NK cells

city: Bethesda

first: Mehdi

last: Pirooznia

state: MD

address: 12 SOUTH DR

institute: National Institutes of Health

data_processing:
base calling by CASAVA (v1.8)
aligned with STAR (v2.5)
gene expression level was determined by HTSeq (v0.6.1)
DEG analysis was done by DESeq2 R package (v2_1.6.3)
Genome_build: hg38
Supplementary_files_format_and_content: xlsx file includes FPKM values for each gene from each sample and zip file includes the DEG analysis outcome

source_name: modified K562-expanded NK cells

growth_protocol: NK cells were isolated from healthy donor buffy coats by ficoll lymphocyte seperation followed by magnetic bead sorting: CD3 depletion and CD56 selection (Mylteni Biotech)

extract_protocol:
RNA was extracted with RNAeasy isolation kit (Qiagen)
RNA libraries were prepared for sequencing using standard Illumina HiSeq protocols by Novogene; 150-bp paired-end reads were generated at a depth of 20X

value: modified K562-expanded NK cells

tag: culture

value: 2 weeks in X-vivo20 (Lonza) +10% heat inactivated human serum + IL-2 ,  37° C

tag: feeder cell population

value: irradiated genetically modified K564


title: Sample 23_Early AD-1

city: Chengdu

first: Tinghua

last: Wang

state: China

address: 17, Section 3 South Renmin Road, Institute of Neurological Disease, Department o

institute: Sichuan University

data_processing:
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: mm9
Supplementary_files_format_and_content: Matrix table with raw gene counts for each sample

source_name: patient with mild AD

growth_protocol: Peripheral Blood Mononuclear Cells (PBMCs) in the blood samples were obtained from normal individuals and AD patients.

extract_protocol:
PBMCs were purified by Ficoll gradient separation, according to the instructions of the manufacturer (Ficoll–PaqueTM Plus, GE Healthcare, Sweden). In short, 3 mL of Ficoll-Paque gradient was pipetted into two 15-ml centrifuge tubes. The blood was diluted with a ratio 1:1 in phosphate-buffered saline (PBS) and carefully layered over the Ficoll-Paque gradient. The tubes were centrifuged for 30 min at 400 × g. The cell interface layer was harvested carefully (8 ml/tube), and the cells were washed twice in PBS (for 10 min at 100 × g). PBMCs were resuspended in 1 mL complete culture medium (DMEM medium supplemented with 10 % newborn calf serum, 1 % PS).
RNA libraries were prepared for sequencing using standard Illumina protocols

value: 71 years old

tag: subject status

value: patient with mild AD

value: blood

value: peripheral blood mononuclear cell


title: K1: modified K562-expanded NK cells

value: irradiated genetically modified K562


title: L6: LCL-expanded NK cells

source_name: LCL-expanded NK cells

value: LCL-expanded NK cells

value: irradiated EBV-infected LCL


title: K2: modified K562-expanded NK cells

value: irradiated genetically modified K563


title: L4: LCL-expanded NK cells


title: L2: LCL-expanded NK cells


title: Sample 28_Early AD-2

value: 73 years old


title: Sample 24_Late AD-1

source_name: patient with severe AD

value: 82 years old

value: patient with severe AD


title: Sample 16_Normal-1

source_name: normal individual

value: 67 years old

value: normal individual


title: Sample 17_Normal-2


title: K6: modified K562-expanded NK cells

value: irradiated genetically modified K567


title: L1: LCL-expanded NK cells


title: L5: LCL-expanded NK cells


title: F5: Peripheral blood NK cells

source_name: Peripheral blood NK cells

value: Peripheral blood NK cells

value: 12hrs in X-vivo20 (Lonza) + 10% heat inactivated human serum, 37° C


title: K5: modified K562-expanded NK cells

value: modified K562-expanded NK cells

value: 2 weeks in X-vivo20 (Lonza) +10% heat inactivated human serum + IL-2 ,  37° C

value: irradiated genetically modified K566


title: F6: Peripheral blood NK cells


title: F4: Peripheral blood NK cells


title: F3: Peripheral blood NK cells


title: Sample 27_Late AD-2

value: 90 years old

value: blood


title: K4: modified K562-expanded NK cells

value: irradiated genetically modified K565


title: F2: Peripheral blood NK cells


title: BT303

value: Tumor

value: WNT


title: F1: Peripheral blood NK cells


title: BT298


title: BT300


title: BT296


title: BT299

value: SHH


title: BT295


title: BT294


title: BT292


title: BT289


title: BT287


title: BT290


title: BT293


title: BT283


title: BT284


title: BT280


title: BT265


title: BT255


title: BT253


title: BT256


title: BT254


title: BT277


title: BT257


title: BT206


title: BT250


title: BT205


title: BT88


title: BT202


title: BT207


title: BT89N

value: para-Tumor

value: n/a (NORM)


title: BT90


title: BT87


title: BT201


title: BT285


title: BT86


title: BT282


title: BT84


title: BT82


title: BT81


title: BT83


title: HX69


title: HX65


title: HX60


title: BT64


title: BT204


title: BT52