title: blood, time point 2, e279

description: Library name: e279_w1


title: blood, time point 1, e293

status: Public on Feb 18 2025

type: SRA

city: Sao Paulo

first: Sintia

last: Belangero

address: Rua Botucatu

department: Morphology and Genetics

country: Brazil

institute: UNIFESP

description: Library name: e293_w0

library_source: transcriptomic

data_processing:
To quantify gene expression, we downloaded the transcript list (manifest file) provided by Illumina. We then converted the target coordinates from the hg19 reference genome to the hg38 reference genome using the liftOver tool. Each target was assigned a unique GENCODE ID by comparing its coordinates with genes annotated by GENCODE [version 38] using the bedtools intersect tool [default parameters]. To guarantee precise GENCODE ID assignment, a manual verification was performed. Other coding genes that overlapped with the targets in the GENCODE annotated transcripts were removed. Kallisto was used to initiate the quantification of target expression. We generated a transcript index based on this new transcript list (excluding overlapping genes) using the command “kallisto index -i”. Kallisto was then executed for each sample individually (single end mode). The analysis considered the average read length calculated for each sample and a standard deviation of 0.0000001. To quantify gene-level expression, the tximport tool was employed.
Assembly: hg38
Supplementary files format and content: raw read counts after tximport

molecule: total RNA

organism: Homo sapiens

source_name: blood

extract_protocol:
Blood samples were collected in PAXgene RNA tubes (PreAnalytix, Switzerland), and leukocyte RNA was extracted from whole blood using the PAXgene Blood RNA Kit (Qiagen, Germany). RNA quality and quantity were assessed with the NanoDropTM 1000 Spectrophotometer (Thermo Fisher Scientific) and the Qubit 2.0 fluorometer with the Qubit RNA BR Assay Kit (Thermo Fisher Scientific), respectively. DNA contamination was verified on a 1.5% agarose gel, and RNA integrity was evaluated using the Agilent 2100 Bioanalyzer and RNA 6000 Nano Kit (Agilent).
Gene expression was measured using next-generation sequencing with the TruSeq® Targeted RNA Expression protocol (Illumina). Genes were selected based on four previous studies, two of them in the Brazilian High Risk Cohort for Mental Conditions (BHRCS). Briefly, 55 genes exhibited differential expression in the blood of BHRCS individuals who transitioned from low to high psychiatric symptoms over a 3- year follow-up period, indicating the onset of symptoms (10.1038/s41398-020-0772-3). Conversely, four genes were differentially expressed in individuals who showed a reduction in psychiatric symptoms, suggesting remission (10.1038/s41398-020-0772-3). Additionally, three genes displayed differential expression in the blood of BHRCS participants with major depressive disorder (10.1016/j.jpsychires.2017.03.015). Furthermore, we included 20 genes known to be differentially expressed in the brains of individuals with autism spectrum disorders, schizophrenia, bipolar disorder, depression, or general mental health conditions (10.1126/science.aad6469), as well as 6 endogenous control genes for blood samples for normalization procedures (10.1186/1755-8794-2-49). A total of 100 ng of RNA was reverse transcribed into cDNA using ProtoScript II Reverse Transcriptase (New England Biolabs). Libraries were prepared using the TruSeq Targeted RNA Expression Kit and the TruSeq® Targeted RNA Custom Oligonucleotide Pool (Illumina). Libraries were pooled and sequenced on the NextSeq 500 instrument (Illumina) using the MidOutput v2 kit (150 cycles) and 100 single end reads.

tag: tissue

value: blood

tag: Sex

value: Female

tag: time

tag: batch


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description: Library name: e384_w0


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description: Library name: e1638_w0


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description: Library name: e1113_w0


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description: Library name: e2004_w1


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description: Library name: e1942_w0


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description: Library name: e2120_w0


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description: Library name: e1251_w0


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description: Library name: e1914_w0


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description: Library name: e1850_w0


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description: Library name: e2028_w0


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description: Library name: e2485_w1


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description: Library name: e1694_w0


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description: Library name: e2082_w1


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description: Library name: e620_w0


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description: Library name: e620_w1


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description: Library name: e712_w1


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description: Library name: e1670_w1


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description: Library name: e3_w0


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description: Library name: e2156_w1


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description: Library name: e1335_w0


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description: Library name: e1990_w0


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description: Library name: e1982_w0


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description: Library name: e1982_w1


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description: Library name: e3_w1


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description: Library name: e196_w1


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description: Library name: e284_w0


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description: Library name: e618_w0


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description: Library name: e618_w1


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description: Library name: e2384_w0


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description: Library name: e1990_w1


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description: Library name: e196_w0


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description: Library name: e418_w1


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description: Library name: e1031_w1


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description: Library name: e2384_w1


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description: Library name: e1031_w0


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description: Library name: e1050_w1


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description: Library name: e1666_w1


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description: Library name: e1975_w1


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description: Library name: e629_w1


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description: Library name: e1499_w0


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description: Library name: e1975_w0


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description: Library name: e1666_w0


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description: Library name: e178_w0


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description: Library name: e1744_w0


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description: Library name: e1670_w0


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description: Library name: e178_w1


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description: Library name: e629_w0


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description: Library name: e1744_w1


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description: Library name: e1050_w0


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description: Library name: e332_w1


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description: Library name: e2006_w1


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description: Library name: e442_w1


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description: Library name: e265_w1


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description: Library name: e2476_w1


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description: Library name: e265_w0


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description: Library name: e73_w1


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description: Library name: e332_w0


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description: Library name: e2006_w0


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description: Library name: e1044_w0


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description: Library name: e1719_w1


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description: Library name: e91_w0


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description: Library name: e73_w0


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description: Library name: e460_w1


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description: Library name: e442_w0


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description: Library name: e1499_w1


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description: Library name: e881_w1


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description: Library name: e1619_w0


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description: Library name: e2118_w0


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description: Library name: e2063_w0


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description: Library name: e1863_w1


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description: Library name: e881_w0


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description: Library name: e750_w0


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description: Library name: e460_w0


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description: Library name: e2290_w0


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description: Library name: e2118_w1


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description: Library name: e1747_w0


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description: Library name: e1619_w1


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description: Library name: e2290_w1


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description: Library name: e397_w0


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description: Library name: e2063_w1


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description: Library name: e1339_w1


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description: Library name: e1258_w1


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description: Library name: e1847_w1


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description: Library name: e1719_w0


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description: Library name: e1747_w1


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description: Library name: e2069_w0


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description: Library name: e1339_w0


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description: Library name: e1258_w0


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description: Library name: e2091_w1


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description: Library name: e935_w0


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description: Library name: e1578_w1


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description: Library name: e1277_w0


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description: Library name: e878_w0


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description: Library name: e1460_w0


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description: Library name: e649_w0


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description: Library name: e1460_w1


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description: Library name: e283_w1


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description: Library name: e1689_w1


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description: Library name: e934_w1


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description: Library name: e1277_w1


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description: Library name: e157_w1


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description: Library name: e1199_w0


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description: Library name: e304_w1


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description: Library name: e1199_w1


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description: Library name: e378_w1


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description: Library name: e1731_w1


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description: Library name: e378_w0


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description: Library name: e1916_w0


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description: Library name: e2073_w1


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description: Library name: e858_w1


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description: Library name: e2497_w1


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description: Library name: e1597_w1


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description: Library name: e858_w0


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description: Library name: e2497_w0


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description: Library name: e1916_w1


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description: Library name: e1597_w0


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description: Library name: e1103_w1


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description: Library name: e92_w1


title: blood, time point 2, e1905

description: Library name: e1905_w1


title: blood, time point 2, e2104

description: Library name: e2104_w1


title: blood, time point 1, e2073

description: Library name: e2073_w0


title: blood, time point 1, e1103

description: Library name: e1103_w0


title: blood, time point 1, e1464

description: Library name: e1464_w0


title: blood, time point 1, e1905

description: Library name: e1905_w0


title: blood, time point 1, e1731

description: Library name: e1731_w0


title: blood, time point 2, e2455

description: Library name: e2455_w1


title: blood, time point 2, e137

description: Library name: e137_w1


title: blood, time point 2, e803

description: Library name: e803_w1


title: blood, time point 2, e1845

description: Library name: e1845_w1


title: blood, time point 2, e388

description: Library name: e388_w1


title: blood, time point 1, e791

description: Library name: e791_w0


title: blood, time point 1, e137

description: Library name: e137_w0


title: blood, time point 1, e2455

description: Library name: e2455_w0


title: blood, time point 1, e2104

description: Library name: e2104_w0


title: blood, time point 1, e388

description: Library name: e388_w0


title: blood, time point 2, e794

description: Library name: e794_w1


title: blood, time point 1, e549

description: Library name: e549_w0


title: blood, time point 1, e2199

description: Library name: e2199_w0


title: blood, time point 2, e791

description: Library name: e791_w1


title: blood, time point 1, e803

description: Library name: e803_w0


title: blood, time point 2, e220

description: Library name: e220_w1


title: blood, time point 2, e549

description: Library name: e549_w1


title: blood, time point 1, e220

description: Library name: e220_w0


title: blood, time point 2, e940

description: Library name: e940_w1


title: blood, time point 1, e2432

description: Library name: e2432_w0


title: blood, time point 1, e794

description: Library name: e794_w0


title: blood, time point 2, e2432

description: Library name: e2432_w1


title: blood, time point 1, e1489

description: Library name: e1489_w0


title: blood, time point 2, e2113

description: Library name: e2113_w1


title: blood, time point 1, e940

description: Library name: e940_w0


title: blood, time point 2, e2199

description: Library name: e2199_w1


title: blood, time point 2, e875

description: Library name: e875_w1


title: blood, time point 1, e2113

description: Library name: e2113_w0


title: blood, time point 1, e1845

description: Library name: e1845_w0


title: blood, time point 1, e875

description: Library name: e875_w0


title: blood, time point 2, e16

description: Library name: e16_w1


title: blood, time point 1, e278

description: Library name: e278_w0


title: blood, time point 2, e1733

description: Library name: e1733_w1


title: blood, time point 1, e1733

description: Library name: e1733_w0


title: blood, time point 2, e1489

description: Library name: e1489_w1


title: blood, time point 1, e694

description: Library name: e694_w0


title: blood, time point 1, e2143

description: Library name: e2143_w0


title: blood, time point 2, e986

description: Library name: e986_w1


title: blood, time point 2, e278

description: Library name: e278_w1


title: blood, time point 2, e2143

description: Library name: e2143_w1


title: blood, time point 1, e986

description: Library name: e986_w0


title: blood, time point 2, e694

description: Library name: e694_w1


title: blood, time point 1, e16

description: Library name: e16_w0


title: blood, time point 1, e7

description: Library name: e7_w0


title: blood, time point 1, e1438

description: Library name: e1438_w0


title: blood, time point 2, e7

description: Library name: e7_w1


title: blood, time point 1, e1324

description: Library name: e1324_w0


title: blood, time point 2, e1556

description: Library name: e1556_w1


title: blood, time point 2, e1521

description: Library name: e1521_w1


title: blood, time point 1, e1556

description: Library name: e1556_w0


title: blood, time point 2, e2405

description: Library name: e2405_w1


title: blood, time point 1, e57

description: Library name: e57_w0


title: blood, time point 2, e1324

description: Library name: e1324_w1


title: blood, time point 2, e57

description: Library name: e57_w1


title: blood, time point 1, e1521

description: Library name: e1521_w0


title: blood, time point 2, e258

description: Library name: e258_w1


title: blood, time point 2, e1505

description: Library name: e1505_w1


title: blood, time point 2, e1438

description: Library name: e1438_w1


title: blood, time point 1, e2405

description: Library name: e2405_w0


title: blood, time point 1, e1505

description: Library name: e1505_w0


title: blood, time point 1, e258

description: Library name: e258_w0


title: blood, time point 2, e336

description: Library name: e336_w1


title: blood, time point 1, e478

description: Library name: e478_w0


title: blood, time point 2, e478

description: Library name: e478_w1


title: blood, time point 2, e2298

description: Library name: e2298_w1


title: blood, time point 2, e1423

description: Library name: e1423_w1


title: blood, time point 1, e336

description: Library name: e336_w0


title: blood, time point 1, e2454

description: Library name: e2454_w0


title: blood, time point 2, e2086

description: Library name: e2086_w1


title: blood, time point 1, e2298

description: Library name: e2298_w0


title: blood, time point 1, e2086

description: Library name: e2086_w0


title: blood, time point 1, e1423

description: Library name: e1423_w0


title: blood, time point 2, e1613

description: Library name: e1613_w1


title: blood, time point 1, e1613

description: Library name: e1613_w0


title: blood, time point 2, e2454

description: Library name: e2454_w1


title: blood, time point 1, e1262

description: Library name: e1262_w0


title: blood, time point 1, e1950

description: Library name: e1950_w0


title: blood, time point 1, e1078

description: Library name: e1078_w0


title: blood, time point 1, e159

description: Library name: e159_w0


title: blood, time point 1, e1701

description: Library name: e1701_w0


title: blood, time point 1, e857

description: Library name: e857_w0


title: blood, time point 1, e1762

description: Library name: e1762_w0


title: blood, time point 2, e1762

description: Library name: e1762_w1


title: blood, time point 2, e857

description: Library name: e857_w1


title: blood, time point 1, e1575

description: Library name: e1575_w0


title: blood, time point 2, e1950

description: Library name: e1950_w1


title: blood, time point 2, e1262

description: Library name: e1262_w1


title: blood, time point 2, e1078

description: Library name: e1078_w1


title: blood, time point 2, e1575

description: Library name: e1575_w1


title: blood, time point 1, e933

description: Library name: e933_w0


title: blood, time point 2, e350

description: Library name: e350_w1


title: blood, time point 2, e159

description: Library name: e159_w1


title: blood, time point 2, e1701

description: Library name: e1701_w1


title: blood, time point 2, e1265

description: Library name: e1265_w1


title: blood, time point 2, e2365

description: Library name: e2365_w1


title: blood, time point 2, e62

description: Library name: e62_w1


title: blood, time point 1, e767

description: Library name: e767_w0


title: blood, time point 2, e1321

description: Library name: e1321_w1


title: blood, time point 1, e1321

description: Library name: e1321_w0


title: blood, time point 1, e1265

description: Library name: e1265_w0


title: blood, time point 1, e350

description: Library name: e350_w0


title: blood, time point 1, e2365

description: Library name: e2365_w0


title: blood, time point 2, e1145

description: Library name: e1145_w1


title: blood, time point 1, e1145

description: Library name: e1145_w0


title: blood, time point 1, e1155

description: Library name: e1155_w0


title: blood, time point 1, e2378

description: Library name: e2378_w0


title: blood, time point 2, e933

description: Library name: e933_w1


title: blood, time point 2, e767

description: Library name: e767_w1


title: blood, time point 1, e54

description: Library name: e54_w0


title: blood, time point 1, e621

description: Library name: e621_w0


title: blood, time point 2, e54

description: Library name: e54_w1


title: blood, time point 2, e688

description: Library name: e688_w1


title: blood, time point 2, e621

description: Library name: e621_w1


title: blood, time point 1, e688

description: Library name: e688_w0


title: blood, time point 1, e256

description: Library name: e256_w0


title: blood, time point 2, e256

description: Library name: e256_w1


title: blood, time point 1, e939

description: Library name: e939_w0


title: blood, time point 2, e1079

description: Library name: e1079_w1


title: blood, time point 2, e2378

description: Library name: e2378_w1


title: blood, time point 2, e939

description: Library name: e939_w1


title: blood, time point 1, e1913

description: Library name: e1913_w0


title: blood, time point 2, e1913

description: Library name: e1913_w1


title: NOTCH3-WT, NOTCH3-R170C, WT6m_brain_male (2 replicates each) and mcao3d

status: Public on Jan 29 2025

city: Guangzhou

first: Chunyi

last: Li

state: Guangdong

address: 600 Tianhe Road

department: Department of Neurology, Mental and Neurological Disease Research Center

country: China

institute: the Third Affiliated Hospital of Sun Yat-sen University

description:
4 samples
10x Genomics

library_source: transcriptomic single cell

data_processing:
Reads were aligned to the mm10 genome using the Cell Ranger software pipeline (version 4.0.0) provided by 10x genomics. The resulting filtered gene by cell matrix of UMI counts was read into R using the read10xCounts function from the Droplet Utils package. Filtering was applied in order to remove low quality cells by excluding cells expressing fewer than 200 or greater than 7,500 unique genes, having fewer than 1,000 or greater than 40,000 UMI counts. Expression values for the remaining cells were then normalized using the scran and scater packages. The resulting log2 values were transformed to the natural log scale for compatibility with the Seurat (v4) pipeline.
Assembly: mm10
Supplementary files format and content: Matrix files

organism: Mus musculus

source_name: Whole brain

extract_protocol:
We used scissors to cut the whole brain into small pieces as much as possible, added 1ml mixed enzyme solution, transfered to a shaker and digested at 200rpm, 37 ° C for 20 minutes. Then we epeatedly blowed the digested tissue homogenate 10 times using a 1ml tip, and transfered the tissue homogenate to the cell filter. The cell filters were rinsed with 6ml of 0.15M physiological saline, then rinsed with 3ml of 100% Percoll to thoroughly remove any remaining brain cells on the filter. The cells were gently mix, and then slowly transfered to a 15ml centrifuge tube containing 2ml of 70% Percoll. Brain cells were centrifuged according to the following parameters: 800g, 18 ° C, increase speed by 3, decrease speed by 3, for 15 minutes. The upper myelin sheath was removed and the liquid is transferred to a clean and sterile 50ml centrifuge tube. After filling with PBS, brain cells were centrifuged at 700g, 4 ° C for 8 minutes. The precipitate obtained was the brain tissue single-cell suspension. Perform cell viability testing on the obtained single-cell suspension, using samples with cell viability greater than 80% and a cell count of 5 * 105 for subsequent experiments.
Single-cell suspensions were loaded onto the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (10X Genomics, 1000414). Sample processing and library preparation was performed according to manufacturer’s instructions.

value: Whole brain

tag: cell type

value: Whole brain cells

tag: strain

tag: age

value: 16 weeks old


title: Nifedipine_D7

status: Public on Jan 15 2025

city: Brisbane

first: James

last: Hudson

state: Queensland

address: 300 Herston Rd, Herston QLD 4006

country: Australia

institute: QIMR Berghofer Medical Research Institute

data_processing:
Sequence reads were aligned using STAR (version 2.7.9a)
Counts matrix was generated using RSEM (version 1.3.1)
Assembly: GRCh38 assembly with the gene, transcript, and exon features of Ensembl (release 106) gene model
Supplementary files format and content: comma separated text file includes raw counts for each sample

extract_protocol:
Total RNA was isolated using the NuceloMag RNA kit (Macherey-Nagel) on the EpMotion 5075t
RNA libraries were prepared using the Illumina Stranded mRNA Prep Ligation kit (polyA RNA selection) on the EpMotion 5075t

treatment_protocol: HES3 hCO were treated with Nifidipine or DMSO (vehicle control) for 8 hours (n = 5 per condition). hCO were washed with PBS, lysed, and stored at -80 degrees.

tag: cell line

tag: treatment

value: Nif


title: blood, time point 2, e1155

description: Library name: e1155_w1


title: blood, time point 1, e62

description: Library name: e62_w0


title: Nifedipine_G9


title: Nifedipine_D12


title: DMSO_B9

value: DMSO


title: DMSO_E10


title: Nifedipine_C1


title: Mouse brain cortex, dscHi-C-multiome, RNA

status: Public on Jan 08 2025

city: Beijing

first: Xiaoliang

middle: Sunney

last: Xie

state: Beijing

address: No.5 Yiheyuan Road, Haidian District

institute: Peking University

description: Library name: b3-month-BC-dscHiC-RNA

library_source: other

data_processing:
dscHiCtools (https://github.com/MaoxuWang/dscHiCtools) firstly identify the true cell barcodes from index reads against whitelist (cellranger-atac-2.1.0/lib/python/atac/barcodes/737K-cratac-v1.txt.gz) barcodes that allows one mismatch. Barcodes with no or multiple hits will be discarded. Called cell barcodes are added to read name. Reads then were mapped to reference genome by bwa-mem in "5SP" mode. Hickit were used to extract and dedup contacts from sam file. Notably, we did minor modifications to Hickit (https://github.com/MaoxuWang/hickit) to adapt the single-cell features. Briefly, we extracted the cell barcode to each contact information and cell barcode was also considered in contacts duplication rather than distance (set parameters --dup-dist=100”) alone. Cells with too few contacts were considered as technique noise and were filtered.
Assembly: mm10 / hg38
Supplementary files format and content: .contacts.pairs.gz includes unique valid contact pairs for each single cell in 4DN format
Supplementary files format and content: .metadata.txt includes basic statistics for each single cell

molecule: other

source_name: Brain cortex

growth_protocol: K562 (ATCC, CCL-243) were cultured in Iscove’s Modified Dulbecco’s medium, BJ (ATCC, CRL-2522) were grown in ATCC-formulated Eagle's Minimum Essential Medium, eHAP (Cellosaurus), engineered haploid chronic myeloid leukemia cell line, were grown in Iscove’s Modified Dulbecco’s Medium, GM12878 (Coriell Institute), B lymphoblastoid cells, were grown in Roswell Park Memorial Institute 1640 Medium. These media were supplemented with 10% FBS and 1% Penicillin/Streptomycin, except for GM12878 supplemented with 15% FBS. mESC were grown in knockout DMEM medium containing 15% FBS, 1% Pen/Strep, 2 mM L-glutamine, 1 mM non-essential amino acids, 1 x nucleosides, 3 μM CHIR99021, 1 μM PD0325901, 0.1 mM 2-mercaptoethanol and 1000 U/mL LIF. The plate was pretreated with 1% gelatin. All cells were maintained at 37°C with 5% CO2 at recommended density.

extract_protocol:
Cultured cell line or isolated nuclei from primary brain cortex were processed with Hi-C 3.0 protocol. Cells were firstly double-crosslinked with PFA followed by DSG, then treated with SDS and Triton X-100 to open chromatin. Then the chromatin was digested by resuspending the nuclei in digestion mix containing DdeI and DpnII without detergent, and incubated at 37°C for 2 hours, followed by inactivating the REs. The DNA ends was filled by biotin labeled dATP, followed by proximity ligation using T4 ligase by incubating at room temperature for 2 hours. For sample prepared for dscHi-C multiome experiment, all reaction mix was supplemented 0.5 U/μL Protector RNase inhibitor.
Nuclei processed through Hi-C 3.0 procedure were bulk tagmented with Tn5 transposome, then loaded to 10x Chromium platform according to either ATAC kit or multiome kit. For dscHi-C, the DNA were barcoded by linear amplification in droplet, followed pooling droplets, further amplification and sample index were incorporated. For dscHi-C-multiome, the DNA and RNA from the same cell were barcoded in droplet, the then droplets were pooled and nuclei were decrosslinked. Genomic DNA were pull down using streptavidin beads, and cDNA were further proceeded with second time reverse transcription. Then the genomic DNA were direct subjected to library preparation. The cDNA were further fragmented and amplified for library construction.

value: Brain cortex

tag: genotype

value: WT


title: DMSO_D6


title: DMSO_A2


title: GSDMD-NT EV-recipient mouse splenocytes, rep2

status: Public on Jan 07 2025

city: Avon

first: Beiyan

last: Zhou

state: Connecticut

address: 263 Farmington Ave, Immunology

department: Immunology

country: USA

institute: UCONN Health

data_processing:
Reads were trimmed by cutadapt/3.5 to remove Illumina adapters
Trimmed reads in Fastq files were aligned using HISAT2 v2.2.1 on the GRCm38 index, with SAMtools v1.9 for BAM conversion.
A matrix of fragments per kilobase of transcript per million fragments mapped (FPKM) was calculated using Stringtie v2.1.3 with Ballgown on the GRCm38 index.
FPKM data was converted to gene count data matrix using tximport v1.32.0 R package
Assembly: GRCm38
Supplementary files format and content: comma separated file including unfiltered gene FPKM
Supplementary files format and content: comma separated file including unfiltered gene counts

molecule: polyA RNA

source_name: Spleen

growth_protocol: in vivo

extract_protocol:
RNA was purified using RNeasy Mini Kit (Qiagen).
RNA libraries were prepared using the Illumina TruSeq Stranded mRNA library prep kit following manufacter's protocols.

treatment_protocol: Gsdmd-/- mice were injected with EVs from pyropototic PBS- or dox-stimulated iBMDM-iGSDMD-NT cells

value: Spleen

value: Splenocytes

value: GSDMD-NT EV


title: Control EV-recipient mouse splenocytes, rep3

value: Control EV


title: GSDMD-NT EV-recipient mouse splenocytes, rep1


title: Control EV-recipient mouse splenocytes, rep2


title: Human CRC NAT  CD45 pos, replicate 7, scRNAseq

status: Public on Dec 17 2024

city: Zürich

first: Kristina

last: Handler

address: Winterthurerstrasse

department: Institute of Experimental Immunology

country: Switzerland

institute: University of Zürich

description: Library name: P7_NAT

data_processing:
Demultiplexing of BCL files with Bcl2fastq (v2.20.0433, Illumina)
Count matrix generation using the BD Rhapsody™ WTA analysis pipeline (v1.12.1) on the SevenBridges Genomics platform
Assembly: STAR index generation using STAR (v2.5.2b) with gene code GRCm38 v25 for mouse and GRCh38 v43 for human data
Supplementary files format and content: Text file showing the input data for the WTA Analysis pipeline at the beginning of the file. Followed by a matrix (first column: “Cell_index”, cell IDs; second column: “Bioproduct”, Gene Symbols; third column: “RSEC_Reads”, Number of UMI reads per cell ID and bioproduct; forth column: “Raw_Molecules”, Reads with the same cell ID, same UMI sequence and same bioproduct collapsed into single molecules; fifth column: “RSEC_Adjusted_Molecules”, Number of molecules detected after RSEC molecular identifier adjustment algorithm, this is the counts used for downstream analysis;

source_name: Normal adjacent tissue (colon)

extract_protocol:
BD Rhapsody Express Single-Cell Analysis System (BD Bioscience)
mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol (BD Biosciences)

treatment_protocol: Cell hasing unsing BD Rhapsody Mouse and Human Single-Cell Multiplexing kit

value: Normal adjacent tissue (colon)

tag: cellular enrichment

tag: bd rhapsody_capture_bead_version

value: enhanced beads

tag: sample tag_(cell_hashing)_version

value: Single-Cell Multiplex Kit - Human

tag: sample tag_(cell_hashing)


title: Nifedipine_B4


title: Human CRC tumor  CD45 pos, replicate 7, scRNAseq

description: Library name: P7_tumor

source_name: Tumor (colon)

value: Tumor (colon)

tag: tumor stage


title: Human CRC NAT  CD45 pos, replicate 6, scRNAseq

description: Library name: P6_NAT

source_name: Normal adjacent tissue (rectum)

value: Normal adjacent tissue (rectum)


title: DMSO_G3


title: Human CRC tumor  CD45 pos, replicate 6, scRNAseq

description: Library name: P6_tumor

source_name: Tumor (rectum)

value: Tumor (rectum)


title: Human CRC NAT  CD45 pos, replicate 5, scRNAseq

description: Library name: P5_NAT


title: Control EV-recipient mouse splenocytes, rep1


title: Human CRC tumor  CD45 pos, replicate 5, scRNAseq

description: Library name: P5_tumor


title: Human CRC NAT  CD45 pos, replicate 3, scRNAseq

description: Library name: P3_NAT


title: Human CRC tumor  CD45 pos, replicate 3, scRNAseq

description: Library name: P3_tumor


title: Human healthy blood eosinophils, replicate 6, scRNAseq

description: Library name: Blood_healthy_6_hs

source_name: Blood

value: Blood

value: Eosinophils


title: Human CRC NAT  CD45 pos, replicate 4, scRNAseq

description: Library name: P4_NAT


title: Human healthy blood eosinophils, replicate 4, scRNAseq

description: Library name: Blood_healthy_4_hs


title: Human healthy blood eosinophils, replicate 3, scRNAseq

description: Library name: Blood_healthy_3_hs


title: Human healthy blood eosinophils, replicate 5, scRNAseq

description: Library name: Blood_healthy_5_hs


title: Human CRC NAT  CD45 pos, replicate 2, scRNAseq

description: Library name: P2_NAT


title: Human healthy blood eosinophils, replicate 1, scRNAseq

description: Library name: Blood_healthy_1_hs


title: Human CRC tumor CD45 pos, replicate 2, scRNAseq

description: Library name: P2_tumor


title: Human healthy blood eosinophils, replicate 2, scRNAseq

description: Library name: Blood_healthy_2_hs


title: GSDMD-NT EV-recipient mouse splenocytes, rep3


title: Human CRC NAT CD45 pos, replicate 1, scRNAseq

description: Library name: P1_NAT


title: Human CRC tumor CD45 pos, replicate 1, scRNAseq

description: Library name: P1_tumor


title: mE14_13hrBrdU_0hrThy_B2_T1

status: Public on Nov 12 2024

city: Los Angeles

first: Marty

middle: G

last: Yang

state: CA

address: 615 Charles E Young Dr S

department: Biological Chemistry

institute: University of California, Los Angeles

description: E14 mESCs were treated with 10 uM BrdU and chased with 10 uM thymidine for varying timepoints to label newly replicated chromatin, nuclei were isolated and treated with EcoGII for footprinting of protein-DNA contacts, and DNA was purified and prepared into PacBio SMRTbell libraries for sequencing on the PacBio Sequel II platform.

library_source: genomic

data_processing:
Reads are demultiplexed using lima to produce a .bam file for subreads for each sample.
Circular consensus sequences (CCS) were generated for each sample using ccs to produce a corresponding .bam file per sample.
Assembly: mm10
Supplementary files format and content: Processed data is shared in .pickle format.
Library strategy: SAMOSA

molecule: genomic DNA

source_name: mESCs

growth_protocol: K562 cells (ATCC) were grown for at least three days in standard media containing RPMI 1640 + L-Glutamine (Hyclone) supplemented with 10% Fetal Bovine Serum (Gibco) and 1% Penicillin-Streptomycin (Gibco). mESC E14 cells were grown on 0.2% gelatin and maintained in standard media containing DMEM + Glutamax (ThermoFisher 10566-016), 15% Fetal Bovine Serum (ThermoFisher SH30071.03), 1X Non-Essential Amino Acids (ThermoFisher 11140-50), 1 mM Sodium Pyruvate (ThermoFisher 11360-070), 0.128mM 2-Mercaptoethanol (BioRad 1610710XTU), and 1X Leukemia Inhibitory Factor (purified and gifted by Barbara Panning Lab at UCSF). mESCs were passaged every 1-2 days using TrypLE Express Enzyme (1X) (12605010). Media was changed daily when cells were not passaged. All cell lines were regularly tested negative for mycoplasma contamination. Standard media was added to mESC E14 or K562 cells with a final concentration of 10 uM BrdU (abcam ab142567). Cells were incubated in BrdU-containing media for between 5 minutes and 24 hours. After labeling, mESC E14 plates were rinsed three times with 1x PBS and collected using TrypLE Express Enzyme (1X) (12605010). K562s were collected and spun down (300xg, 5 minutes) immediately after labeling.

extract_protocol:
To the methylation reaction, 2.65 uL 10% SDS and 2.65 uL of 20 mg/mL Proteinase K (Thermo Scientific AM2548) were added and incubated at 65oC for at least 2 hours and up to overnight. To extract the DNA, an equal volume of Phenol-Chloroform was added and mixed vigorously by shaking. The samples were then spun at max speed for 2 min and the aqueous portion was removed. To the aqueous extraction, 0.1x volumes of 3M NaOAc, 1 uL GlycoBlue, and 3x volumes of cold 100% EtOH were added, mixed by inversion and incubated overnight at -20oC or for 2 hours at -80oC. Samples were then spun at max speed for 30 minutes at 4oC, washed with 500 uL 70% EtOH, spun again at max speed for 2 minutes at 4oC. The resulting pellet was air dried and resuspended in 40 uL EB.
Purified DNA from mESCs and K562s was sheared using a Covaris g-tube (520079) in a 5424 rotor at 7,000 RPM for 6 passes for a target size between 6,000 and 8,000 bp. Sheared DNA was used as input for the PacBio HiFi SMRTbell Library Preparation protocol using the SMRTbell Express Template Prep Kit 2.0. Briefly, samples underwent removal of single stranded overhangs, DNA damage repair, end-repair, A-tailing, barcoded SMRTbell adapter ligation, and exonuclease cleanup according to manufacturer’s protocol followed by a 1X AMPure PB bead cleanup. Final sample concentration was measured via Qubit High Sensitivity DNA Assay and library size was measured on an Agilent Bioanalyzer DNA chip. Libraries were sequenced on PacBio Sequel II 8M SMRTcells.

treatment_protocol: Briefly, all nuclei were collected by centrifugation (300xg, 5 min), washed in ice cold 1X PBS, spun again (300xg, 5 min), and resuspended in 1 mL Nuclear Isolation Buffer (20mM HEPES, 10mM KCl, 1mM MgCl2, 0.1% Triton X-100, 20% Glycerol, and 1X Protease Inhibitor (Roche 4693132001)) per 10 million cells by gently pipetting 5x with a wide-bore pipette tip. The suspension was incubated on ice for 5 minutes, and nuclei were pelleted (600xg, 4oC, 5 min), washed with Buffer M (15mM Tris-HCl pH 8.0, 15 mM NaCl, 60mM KCl, 0.5mM Spermidine), and spun once again (600xg, 4oC, 5 min). Nuclei were resuspended in 200 uL of Methylation Reaction Buffer (Buffer M containing 1mM S-adenosylmethionine (SAM; New England BioLabs B9003S)). 10uL high-concentration EcoGII was added per 1e6 nuclei and the nuclei suspension was incubated at 37oC for 30 minutes. An additional 1 uL of 32 mM SAM was supplemented after 15 minutes. Any unmethylated controls were treated similarly with the addition of SAM, but EcoGII was excluded from those reactions.

value: mESCs

value: EcoGII treated; 13hr BrdU; no Thy


title: mE14_1hrBrdU_12hrThy_B1_T1

value: EcoGII treated; 1hr BrdU; 12hr Thy


title: mE14_6hrBrdU_0hrThy_B1_T1

value: EcoGII treated; 6hr BrdU; no Thy


title: mE14_13hrBrdU_0hrThy_B1_T1


title: mE14_3hrBrdU_0hrThy_B2_T1

value: EcoGII treated; 3hr BrdU; no Thy


title: mE14_3hrBrdU_0hrThy_B1_T1


title: Human CRC tumor  CD45 pos, replicate 4, scRNAseq

description: Library name: P4_tumor


title: mE14_1hrBrdU_2hrThy_B2_T1

value: EcoGII treated; 1hr BrdU; 2hr Thy


title: mE14_1hrBrdU_2hrThy_B1_T1


title: mE14_1hrBrdU_0hrThy_B1_T1

value: EcoGII treated; 1hr BrdU; no Thy


title: noME14_10m_CsSO4_Gradient_B2_T1

description: E14 mESCs were treated with 10 uM BrdU for varying timepoints to label newly replicated chromatin, nuclei were isolated and treated with EcoGII for footprinting of protein-DNA contacts, and DNA was purified, fractionated by CsSO4 density equilibrium gradient centrifugation, and prepared into PacBio SMRTbell libraries for sequencing on the PacBio Sequel II platform.

value: no EcoGII; 10m BrdU; post-CsSO4 gradient


title: noME14_60m_CsSO4_Gradient_B2_T1

value: no EcoGII; 60m BrdU; post-CsSO4 gradient


title: noME14_60m_CsSO4_Input_B2_T1

value: no EcoGII; 60m BrdU; pre-CsSO4 gradient


title: mE14_60m_CsSO4_Gradient_B1_T1

value: EcoGII treated; 60m BrdU; post-CsSO4 gradient


title: mE14_60m_CsSO4_Input_B1_T1

value: EcoGII treated; 60m BrdU; pre-CsSO4 gradient


title: mE14_10m_CsSO4_Gradient_B1_T1

value: EcoGII treated; 10m BrdU; post-CsSO4 gradient


title: CAF1degronPlus_60m_B2_T2

description: CAF-1-dTAG mESCs were provided by Anja Groth's lab (University of Copenhagen). Cells were treated with 0.5 uM dTAG-V1 or DMSO for 2h prior to any BrdU labeling, after which nuclei were isolated and treated with EcoGII for footprinting of protein-DNA contacts, and DNA was purified and prepared into PacBio SMRTbell libraries for sequencing on the PacBio Sequel II platform.

value: EcoGII treated; 2hr dTAG-V1 + 1hr BrdU


title: CAF1degronPlus_10m_B2_T2

value: EcoGII treated; 2hr dTAG-V1 + 10m BrdU


title: CAF1degronMinus_60m_B2_T2

value: EcoGII treated; 2hr DMSO + 1hr BrdU


title: CAF1degronMinus_15m_B2_T2

value: EcoGII treated; 2hr DMSO + 15m BrdU


title: mE14_1hrBrdU_5hrThy_B1_T1

value: mESCs

value: EcoGII treated; 1hr BrdU; 5hr Thy


title: CAF1degronPlus_60m_B1_T3


title: mE14_1hrBrdU_5hrThy_B2_T1


title: mE14_6hrBrdU_0hrThy_B2_T1


title: CAF1degronPlus_10m_B1_T3


title: CAF1degronMinus_10m_B2_T2

value: EcoGII treated; 2hr DMSO + 10m BrdU


title: mE14_1hrBrdU_12hrThy_B2_T1

value: EcoGII treated; 1hr BrdU; 12hr Thy


title: CAF1degronMinus_60m_B1_T3


title: CAF1degronMinus_10m_B1_T3


title: CAF1degronPlus_60m_B1_T2


title: CAF1degronMinus_15m_B1_T3


title: CAF1degronPlus_15m_B1_T2

value: EcoGII treated; 2hr dTAG-V1 + 15m BrdU


title: CAF1degronMinus_60m_B1_T2


title: noME14_10m_CsSO4_Input_B2_T1

value: no EcoGII; 10m BrdU; pre-CsSO4 gradient


title: CAF1degronMinus_15m_B1_T2


title: CAF1degronPlus_10m_B1_T2


title: CAF1degronPlus_15m_B2_T1


title: CAF1degronMinus_10m_B1_T2


title: CAF1degronPlus_60m_B2_T1


title: CAF1degronPlus_15m_B2_T2


title: mE14_10m_CsSO4_Input_B1_T1

value: EcoGII treated; 10m BrdU; pre-CsSO4 gradient


title: CAF1degronMinus_60m_B2_T1


title: CAF1degronMinus_15m_B2_T1


title: CAF1degronPlus_10m_B2_T1


title: CAF1degronPlus_15m_B1_T1


title: CAF1degronMinus_10m_B2_T1


title: CAF1degronPlus_10m_B1_T1


title: CAF1degronPlus_15m_B1_T3


title: CAF1degronMinus_15m_B1_T1


title: CAF1degronMinus_60m_B1_T1


title: CTCFdegronPlus_360m_B1

description: CTCF-AID mESCs were provided by Elphege Nora's lab (UCSF). Cells were treated with 0.5 mM auxin or DMSO for 6h prior to any BrdU labeling, after which nuclei were isolated and treated with EcoGII for footprinting of protein-DNA contacts, and DNA was purified and prepared into PacBio SMRTbell libraries for sequencing on the PacBio Sequel II platform.

value: CTCF-AID

value: EcoGII treated; 6hr DMSO + 6hr BrdU


title: CTCFdegronMinus_360m_B2

value: EcoGII treated; 6hr auxin + 6hr BrdU


title: CTCFdegronMinus_360m_B1


title: CTCFdegronPlus_360m_B2


title: noME14_1440m_B1_T1

description: E14 mESCs were treated with 10 uM BrdU for varying timepoints to label newly replicated chromatin, nuclei were isolated and treated with EcoGII for footprinting of protein-DNA contacts, and DNA was purified and prepared into PacBio SMRTbell libraries for sequencing on the PacBio Sequel II platform.

data_processing:
Reads are demultiplexed using lima to produce a .bam file for subreads for each sample.
Circular consensus sequences (CCS) were generated for each sample using ccs to produce a corresponding .bam file per sample.
Supplementary files format and content: Processed data is shared in .pickle format.
Library strategy: Replication-Aware Single-molecule Accessibility Mapping (RASAM)

value: no EcoGII; 24h BrdU


title: mE14_720m_B2_T4

value: EcoGII treated; 12h BrdU


title: noME14_5m_B1_T1

value: no EcoGII; 5m BrdU


title: mE14_720m_B2_T3


title: noME14_0m_B1_T1

value: no EcoGII; no BrdU


title: mE14_720m_B2_T2


title: mE14_720m_B2_T1


title: mE14_720m_B1_T4


title: mE14_720m_B1_T3


title: mE14_720m_B1_T1


title: CAF1degronPlus_60m_B1_T1

value: EcoGII treated; 2hr dTAG-V1 + 1hr BrdU


title: mE14_60m_B3_T4

value: EcoGII treated; 1h BrdU


title: mE14_60m_B3_T1


title: mE14_60m_B3_T2


title: mE14_60m_B3_T3


title: mE14_60m_B2_T4


title: mE14_60m_B2_T3


title: mE14_60m_B2_T1


title: mE14_60m_B2_T2


title: CAF1degronMinus_10m_B1_T1


title: mE14_60m_B1_T2


title: mE14_5m_B3_T4

value: EcoGII treated; 5m BrdU


title: mE14_5m_B3_T3

value: mESCs


title: mE14_5m_B3_T1


title: mE14_60m_B1_T1


title: mE14_5m_B2_T4


title: noME14_15m_B1_T1

value: no EcoGII; 15m BrdU


title: mE14_5m_B2_T1


title: mE14_5m_B2_T2


title: mE14_5m_B1_T3


title: mE14_720m_B1_T2


title: mE14_5m_B1_T2


title: mE14_5m_B1_T1


title: mE14_360m_B3_T4

value: EcoGII treated; 6h BrdU


title: mE14_360m_B3_T3


title: mE14_360m_B3_T2


title: mE14_360m_B2_T4


title: mE14_360m_B2_T2


title: mE14_360m_B2_T3


title: mE14_360m_B2_T1


title: mE14_360m_B1_T2


title: mE14_360m_B1_T1


title: mE14_15m_B3_T3

value: EcoGII treated; 15m BrdU


title: mE14_5m_B3_T2


title: mE14_15m_B2_T4


title: mE14_15m_B3_T1


title: mE14_5m_B2_T3


title: mE14_15m_B2_T2


title: mE14_15m_B2_T3


title: mE14_15m_B1_T3


title: mE14_15m_B2_T1


title: mE14_15m_B1_T2


title: mE14_1440m_B2_T1

value: EcoGII treated; 24h BrdU


title: mE14_1440m_B1_T3


title: mE14_1440m_B1_T2


title: mE14_15m_B1_T1


title: mE14_1440m_B1_T1


title: mE14_10m_B3_T2

value: EcoGII treated; 10m BrdU


title: mE14_360m_B3_T1


title: mE14_10m_B3_T3


title: mE14_10m_B2_T4


title: mE14_15m_B3_T4


title: mE14_10m_B2_T3


title: mE14_10m_B3_T1


title: mE14_10m_B2_T1


title: mE14_10m_B2_T2


title: mE14_0m_B1_T1

value: EcoGII treated; no BrdU


title: mE14_10m_B1_T1


title: mE14_15m_B3_T2


title: CTCFdegronPlus_60m_B2_T2

description: CTCF-AID mESCs were provided by Elphege Nora's lab (UCSF). Cells were treated with 0.5 mM auxin for 6h prior to any BrdU labeling, after which nuclei were isolated and treated with EcoGII for footprinting of protein-DNA contacts, and DNA was purified and prepared into PacBio SMRTbell libraries for sequencing on the PacBio Sequel II platform.

value: CTCF-AID

value: EcoGII treated; 60m BrdU; auxin treated


title: CTCFdegronPlus_60m_B2_T1


title: CTCFdegronPlus_60m_B1_T2


title: CTCFdegronPlus_60m_B1_T1


title: mE14_1hrBrdU_0hrThy_B2_T1

value: EcoGII treated; 1hr BrdU; no Thy


title: CTCFdegronPlus_15m_B2_T1

value: EcoGII treated; 15m BrdU; auxin treated


title: CTCFdegronPlus_15m_B1_T2


title: CTCFdegronPlus_15m_B1_T1


title: CTCFdegronPlus_15m_B2_T2


title: CTCFdegronPlus_10m_B2_T1

value: EcoGII treated; 10m BrdU; auxin treated


title: CTCFdegronPlus_10m_B2_T2


title: mE14_10m_B3_T4


title: CTCFdegronPlus_10m_B1_T1


title: CTCFdegronMinus_60m_B1_T2

value: EcoGII treated; 60m BrdU


title: CTCFdegronMinus_60m_B1_T1


title: CTCFdegronMinus_60m_B2_T2


title: CTCFdegronMinus_15m_B1_T2


title: CTCFdegronMinus_15m_B2_T1


title: CTCFdegronMinus_15m_B1_T1


title: CTCFdegronMinus_10m_B2_T2


title: CTCFdegronMinus_10m_B2_T1


title: CTCFdegronMinus_10m_B1_T2


title: CTCFdegronMinus_10m_B1_T1


title: noMK562_10m_B1_T1

description: K562 cells were treated with 10 uM BrdU for varying timepoints to label newly replicated chromatin, nuclei were isolated and treated with EcoGII for footprinting of protein-DNA contacts, and DNA was purified and prepared into PacBio SMRTbell libraries for sequencing on the PacBio Sequel II platform.

value: no EcoGII; 10m BrdU


title: noMK562_0m_B1_T1

value: no EcoGII; no BrdU


title: mK562_360m_B2_T1


title: noMK562_120m_B1_T1

value: no EcoGII; 120m BrdU


title: noMK562_1440m_B1_T1

value: no EcoGII; 1440m BrdU


title: mK562_360m_B1_T1


title: mK562_1440m_B2_T2


title: mK562_1440m_B2_T1


title: CTCFdegronPlus_10m_B1_T2

value: mESCs


title: mK562_1440m_B1_T1


title: CTCFdegronMinus_60m_B2_T1


title: mK562_120m_B1_T1

value: EcoGII treated; 2h BrdU


title: mK562_120m_B2_T1


title: mK562_10m_B1_T1


title: CTCFdegronMinus_15m_B2_T2


title: mK562_0m_B2_T2


title: mK562_0m_B1_T1


title: mK562_0m_B2_T1


title: Hela cells expressing 3×Flag-TFEB, Ctrl, rep3

status: Public on Nov 10 2024

city: Avenida da Universidade Taipa

first: Chen

last: Ming

address: Macau

department: Faculty of Health Sciences

country: Macau

institute: the University of Macau

description: gene.expression.txt

data_processing:
Three replicates of paired-end reads were trimmed and filtered by Fastp and qualified by FastQC, reads were aligned to the hg38 RefSeq database using HISAT2.
The gene count matrix and transcript count matrix were generated by Stringtie.
Genes with FPKM (fragments per kilobase per million mapped reads) larger than or equal to 0.1 were included.
Differential gene expression was performed using DESeq2.
Differential expression genes (DEGs) were selected based on corrected p-value using the Benjamini-Hochberg method (Padj) <0.05 and fold change (FC) > 1.5 or < -1.5.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include raw counts for each sample
Supplementary files format and content: tab-delimited text files include DEG results

source_name: Hela cells

growth_protocol: Cells were cultured using DMEM containing 10% FBS and 1% PS

extract_protocol:
For RNA-seq, total RNA was extracted from Hela cells expressing 3×Flag-TFEB using RNA stabilization and storage solution (RNAlater™, Thermo, AM7020)
For RNA-seq, the RNA extraction was followed by reverse transcription with an oligo-dT primer, RNA library preparation, and paired-end sequencing was performed at the DNBSeq platform by Bioyi Biotechnology Co., Ltd. Wuhan.

treatment_protocol: CCCP (20µM) for 6h; sucrose (150mM) for 8h; Torin1 (1µM) for 6h

value: Hela cells

value: Cervical cancer cells

value: expressing 3xFlag-TFEB

value: Non


title: Hela cells expressing 3×Flag-TFEB, Ctrl, rep2

description: gene.expression.txt


title: Hela cells expressing 3×Flag-TFEB, Ctrl, rep1

description: gene.expression.txt


title: mK562_360m_B2_T2

value: EcoGII treated; 6h BrdU


title: Section_21

status: Public on Nov 04 2024

type: RNA

city: Memphis

first: Jasmine

last: Plummer

state: TN

address: 262 Danny Thomas Pl

department: Developmental Neurobiology

institute: St. Jude Children's Research Hospital

description: Fresh Frozen

hyb_protocol: Samples in this study were processed following the guidelines outlined in the CosMx SMI Manual Slide Preparation for RNA Assays user manual (MAN-10184-02, NanoString). Fresh frozen (FF) mouse embryo samples were cut to 10 µm thickness and placed on a Superfrost Plus Premium Microscope Slide (VWR, 48311-703). Slides were stored at -80°C until they were submerged in 10% neutral buffered formalin (NBF) (EMS Diasum, 15740) at 4°C for two hours. Slides were then washed 3 times in 1X PBS (ThermoFisher, AM9625, AM9922) for two minutes each, then baked vertically at 60°C for 30 minutes. Once the tissue was fixed to the slide, fresh frozen samples were washed three times in 1X PBS for five minutes, one time in 4% SDS (ThermoFisher, AM9822) for two minutes, and three times in 1X PBS for 5 minutes each. Slides were then rehydrated with a 50% ethanol wash, a 70% ethanol wash, and two 100% ethanol washes for five minutes each. Next, samples were incubated in 1X Target Retrieval Solution (NanoString CosMx FF Slide Preparation Kit, RNA) at 100°C in a steamer for 8 minutes for heat induced epitope retrieval (HIER). Immediately following HIER, slides were dipped in DEPC-treated water (ThermoFisher, AM9922) for 15 seconds, washed in 100% ethanol for 3 minutes, and subsequently left to dry on the benchtop on a clean Kimwipe at room temperature for 30 minutes. After drying, the tissue was incubated at room temperature with 400 µL of digestion buffer, which contained a combination of Proteinase K and Protease A (NanoString CosMx FF Slide Preparation Kit, RNA), for 15 minutes followed by two 1X PBS washes for five minutes each. Next, slides were incubated at room temperature with fiducials (NanoString CosMx FF Slide Preparation Kit, RNA) at a working concentration of 0.00015% for five minutes protected from light, followed by one five-minute 1X PBS wash to remove excess fiducials. Slides then underwent a 1-minute incubation in 10% NBF followed by two, five-minute NBF STOP buffer washes, and one five-minute 1X PBS wash. A 100 mM NHS-Acetate mixture was prepared using Sulfo NHS-Acetate powder (Fisher Scientific, 26777) and NHS-Acetate buffer (NanoString CosMx FF Slide preparation Kit, RNA) which was then applied to the tissue and left to incubate covered from light at room temperature for 15 minutes. The slides were washed in 2X saline sodium citrate (SSC) (ThermoFisher, AM9763, AM9922) twice for 5 minutes each after NHS-acetate incubation.  The CosMx Mouse Universal Cell Characterization panel contains 950 mouse gene targets with an additional set of 50 custom targets, chosen specifically for this study. The NanoString and custom ISH probes were denatured at 95°C for two minutes and immediately transferred to ice for one minute. The ISH probes were then combined with RNase inhibitor, Buffer R, and DEPC-treated water and applied to the slides as they were placed one-by-one into a hybridization chamber. Coverslips were applied to the slides to prevent loss of the probe mix during overnight hybridization. The slides were incubated overnight for 17 hours protected from light at 37°C. After overnight probe hybridization, slides were washed in 2X SSC to remove coverslips and subsequently washed twice in a 50% formamide (ThermoFisher, AM9342) solution with 2X SSC for 25 minutes at 37°C to remove off target probes. Formamide washes were followed by two 2X SSC washes for 2 minutes each at room temperature. The DAPI stock solution (NanoString, CosMx Mouse Universal Cell Segmentation Kit) was diluted 1:40 and applied to each slide for 15 minutes at room temperature. Following nuclear staining, slides were washed in 1X PBS for 5 minutes before a cocktail of antibodies including CD298/B2M, PanCK, and CD45 (NanoString, CosMx Mouse Universal Cell Segmentation Kit) was applied to tissue for 1 hour at room temperature. Slides were then washed in 1X PBS for 5 minutes for a total of 3 washes to remove off target antibodies and stored overnight at 4°C in 2X SSC.

scan_protocol: A CosMx flow cell was applied to each slide using the CosMx flow cell tool, which involves carefully drying the slide around the tissue, placing the slide in the CosMx flow cell tool, applying the CosMx flow cell, and closing the flow cell tool to fully adhere the flow cell to the slide. Once the flow cell is on the slide, 200 microliters of 2X SSC are pipetted into the flow cell to keep the tissue from drying. Slides were then loaded onto the CosMx SMI per manual MAN-10161-05, the CosMx SMI Instrument User Manual, using pre-bleaching profile C and cell segmentation profile A. CosMx instrument buffer bottles 1-4 were prepared by adding pyranose oxidase and catalase to bottle 4, and each bottle is loaded onto the CosMx instrument. Preview scans of each slide were taken by the CosMx SMI, and fields of view (FOVs) were selected to encompass the entirety of each embryo.

data_processing:
Using the AtoMx platform, images were segmented to obtain cell boundaries, assign transcripts at the cell level, and generate a transcript-by-cell count matrix. This process involved removing unwanted probes and filtering out low-quality cells. First, control and system probes were eliminated, followed by the retention of only high-quality cells based on specific criteria related to total counts, number of features, and cell area (greater than 10, greater than 10, and less than 500 µm², respectively). All downstream analyses were conducted using the Seurat-based workflow in R
Processed data: Transcript counts derived from intensity values within the boundaries of segmented cells exported from AtoMx plaftform

label: SMI imaging barcodes

label_protocol: CosMx barcoded probes for RNA assay with 950 standared CosMx probes and 50 custom add-on CosMx RNA probes.

extract_protocol: Test samples at 10µm thickness were extracted using the Qiagen Rneasy MinElute Cleanup kit (74204) and tested using the TapeStation HS RNA kit (Kit components: 5067-5579, 5067-5580, and 5067-5581) to ensure RNA quality before the start of the CosMx run.

treatment_protocol: Knockout and wild-type samples used for RNA profiling on the CosMx platform as well as Hematoxylin and Eosin (H&E) tissue staining. Tissue was treated with 10% Neutral buffered formalin (NBF) and embedded in paraffin (FFPE).

tag: sample source

value: St. Jude Children's Research Hospital

value: Embryo

tag: technology

value: CosMx

value: Wildtype

tag: panel

tag: custom panel


title: Section_16

description: Fresh Frozen

tag: tissue


title: Section_19

description: Fresh Frozen


title: Section_20

description: Fresh Frozen


title: Section_17

description: Fresh Frozen


title: Section_18

description: Fresh Frozen


title: Section_14

description: Fresh Frozen


title: Section_15

description: Fresh Frozen


title: Section_13

description: Fresh Frozen

value: Knockout


title: Section_10

description: Fresh Frozen


title: Section_9

description: Fresh Frozen


title: Section_8

description: Fresh Frozen


title: mK562_10m_B2_T2

value: EcoGII treated; 10m BrdU


title: Section_12

description: Fresh Frozen


title: Section_11

description: Fresh Frozen


title: mK562_10m_B2_T1


title: Section_6

description: Fresh Frozen


title: mK562_120m_B2_T2


title: Section_7

description: Fresh Frozen


title: Section_4

description: Fresh Frozen


title: Section_3

description: Fresh Frozen


title: Section_5

description: Fresh Frozen


title: Sublibary 8

status: Public on Nov 01 2024

city: Richardson

first: Catherine

middle: A

last: Thorn

state: TX

address: 800 W Campbell Rd

department: Neuroscience

institute: The University of Texas at Dallas

data_processing:
Data was preprocessed using the Parse Biosciences Cloud-Based Pipeline to obtain cell-gene count matrices for each replicate group. Sequencing data was then analyzed with R (version 4.3.2) using Seurat (version 4.1.3).
Assembly: mRatBN7.2
Supplementary files format and content: Gene list
Supplementary files format and content: Cell metadata matching barcodes to original samples (right/left nodose ganglion, male/female) and gene and read counts for every cell

molecule: nuclear RNA

organism: Rattus norvegicus

source_name: nodose ganglia

extract_protocol:
The right and left NG of experimentally naïve, 8-week-old male (n=18) and female (n=18) rats were dissected. rats were deeply anesthetized with 5% isoflurane and sacrificed via rapid decapitation. The right and left NG were dissected and stored in separate PCR tubes before flash-freezing on dry ice. The first side of dissection was counterbalanced across rats. NG samples were stored in a -80 ⁰C freezer until sample preparation.
Individual NG were pooled to prepare three replicate groups for each of the four treatment groups (Male|Left; Male|Right; Female|Left; Female|Right). Each replicate group was created by pooling the right or left NG samples from 6 rats. Nuclei were fixed and barcoded according to Parse Biosciences protocols (Evercode Version 2 Nuclei Fixation Kit, Version 2 WT Kit). The samples were split into 8 sublibraries and were sequenced via paired-end sequencing using the Illumina Nextseq 2000 at the Genome Center at the University of Texas at Dallas.

value: nodose ganglia

value: Long Evans

value: 8 weeks old


title: Section_1

description: Fresh Frozen


title: Sublibary 7


title: Sublibary 5


title: Sublibary 6


title: Section_22

description: Fresh Frozen


title: Sublibary 4


title: Sublibary 3


title: KpnDRT2_WT_uninfected_Term-seq

status: Public on Aug 11 2024

city: New York

first: Samuel

last: Sternberg

address: 701 W. 168th Street, HHSC 726

institute: Columbia University

data_processing:
Datasets were processed using cutadapt (v4.2) to remove Illumina adapter sequences, trim low-quality ends from reads, and filter out reads shorter than 15 bp. Reads were mapped to a combined reference file containing the MG1655 genome (NC_000913.3) and pSL4878 plasmid sequence (see Tang et al., 2024, Table S3), using bowtie2 with default parameters. SAMtools (v1.17) was used to sort and index alignments.
dRNA-seq alignment coordinates were extracted from read 1 using the bamtobed utility from bedtools (v2.31.0). The 5′ boundary of each read was determined as the start coordinate of read 1, for transcripts on the top strand, or the end coordinate of read 1, for transcripts on the bottom strand. The 5′ boundary thus defined for each read was plotted as a histogram over the KpnDRT2 ncRNA locus. Transcription start sites were evaluated based on enrichment of a given position in the Terminator Exonuclease-treated sample compared to the untreated control.  Term-seq alignment coordinates were extracted from read 2 using the bamtobed utility from bedtools (v2.31.0). The 3′ boundary of each read was determined as the end coordinate of read 2, for transcripts on the top strand, or the start coordinate of read 2, for transcripts on the bottom strand. RNA 3′ ends were plotted as a histogram over the KpnDRT2 ncRNA locus.
Assembly: The E. coli MG1655 genome reference sequences can be found in the NCBI database via the accession number NC_000913.3. The pSL4878 plasmid reference sequence can be found in Tang et al. (2024) Table S3.
Supplementary files format and content: BED (.bed) files contain the alignment coordinates for read 1 from dRNA-seq experiments and read 2 from Term-seq experiments.

organism: Escherichia coli str. K-12 substr. MG1655

growth_protocol: E. coli str. K-12 substr. MG1655 (sSL0810) was transformed with plasmids encoding KpnDRT2 and its native flanking sequence. Individual colonies were inoculated in liquid LB with chloramphenicol and grown at 37 °C to OD600 of 0.5.

extract_protocol:
Total RNA was extracted using TRIzol (Thermo Fisher Scientific).
For dRNA-seq library preparation, 1 µg of total RNA was fragmented in 1× NEBuffer 2 by heating at 92 °C for 1.5 min. DNase treatment was performed with 1 μL TURBO DNase (Thermo Fisher Scientific) in the presence of 1 μL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) for 10 min at 37 °C. Samples were treated with 1 μL T4 PNK in 1× T4 DNA ligase buffer (NEB) at 37 °C for 30 min and purified using the Zymo RNA Clean & Concentrator-5 kit. Samples were split in two and either treated to enrich primary transcripts or used as untreated controls. To enrich primary transcripts with tri-phosphorylated 5′ ends, samples were treated with 1 μL of Terminator Exonuclease (Biosearch Technologies) in 1× Terminator Reaction Buffer A (Biosearch Technologies) supplemented with 0.5 μL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). Reactions were incubated at 30 °C for 1 hour and stopped by adding EDTA to a final concentration of 5 mM. Samples were purified using the Zymo RNA Clean & Concentrator-5 kit, and then treated with 2 μL RppH (NEB) in 1× NEBuffer 2 supplemented with 1 μL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). Reactions were incubated at 37 °C for 30 min and purified using the Zymo RNA Clean & Concentrator-5 kit. Illumina sequencing libraries were prepared using the NEBNext Small RNA Library Prep kit.  For Term-seq library preparation, 1 µg of total RNA was treated with 1 µL TURBO DNase in 1× TURBO DNase Buffer (Thermo Fisher Scientific) supplemented with 1 µL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) for 10 min at 37 °C, followed by cleanup using the Zymo RNA Clean & Concentrator-5 kit. Ligation of an i7 Illumina adapter to RNA 3′ ends was performed using the NEBNext Small RNA Library Prep kit, followed by cleanup using the Zymo RNA Clean & Concentrator-5 kit. Samples were fragmented in 1× NEBuffer 2 by heating at 92 °C for 1.5 min, then treated with 2 μL RppH (NEB) in the presence of 1 μL SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) for 30 min at 37 °C. This was followed by treatment with 1 μL T4 PNK in 1× T4 DNA ligase buffer (NEB) at 37 °C for 30 min and cleanup using the Zymo RNA Clean & Concentrator-5 kit. Illumina library preparation continued with the remainder of the NEBNext Small RNA Library Prep protocol after the initial i7 adapter ligation step.  Libraries were sequenced on an Illumina NextSeq 500 in paired-end mode with 75 cycles per end.


title: Sublibary 2


title: KpnDRT2_SL2_MUT_RIP

data_processing:
RIP-seq and corresponding input datasets were processed using cutadapt (v4.2) to remove Illumina adapter sequences, trim low-quality ends from reads, and filter out reads shorter than 15 bp. Reads were mapped to combined reference files containing the MG1655 genome (NC_000913.3) and relevant plasmid sequence, as well as the T5 genome (NC_005859.1) for +/- infection experiments, using bwa-mem2 (v2.2.1) with default parameters. SAMtools (v1.17) was used to sort and index alignments. Coverage tracks were generated using bamCoverage (v3.5.1) with a bin size of 1, separation of top and bottom strand alignments, and scaling of coverage according to sequencing depth (based on the total number of reads passing initial trimming and length filtering).
Assembly: Genome reference sequences can be found in the NCBI database via the accession numbers NC_000913.3 (E. coli MG1655) and NC_005859.1 (phage T5). Plasmid reference sequences can be found in Tang et al. (2024) Table S3.
Supplementary files format and content: bigWig (.bw) files represent normalized read coverage files, separated by strand.

growth_protocol: E. coli str. K-12 substr. MG1655 (sSL0810) was transformed with plasmids encoding C-terminally 3×FLAG-tagged Retron-Eco1 or N-terminally 3×FLAG-tagged KpnDRT2 (WT, RT mutant, or ncRNA mutant), as well as their native flanking sequences. Individual colonies were inoculated in liquid LB with chloramphenicol and grown at 37 °C to OD600 of 0.5.

extract_protocol:
Cells were harvested by centrifugation at 4,000 x g for 10 min at 4 °C, and the supernatant was removed. The pellet was washed with 5 mL of cold TBS (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM NaCl) and spun down again as before. The supernatant was removed, and the pellet was washed with 1 mL of cold TBS before centrifugation at 10,000 x g for 5 min at 4 °C. The supernatant was removed, and the pellet was flash-frozen in liquid nitrogen and stored at -80 °C. Antibodies for immunoprecipitation were conjugated to magnetic beads as follows: for each sample, 60 μL Dynabeads Protein G (Thermo Fisher Scientific) were washed 3× in 1 mL IP lysis buffer (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM KCl, 1 mM MgCl2, 0.2% Triton X-100), resuspended in 1 mL IP lysis buffer, combined with 20 μL anti-FLAG M2 antibody (Sigma-Aldrich, F3165), and rotated for > 3 hours at 4 °C. Antibody-bead complexes were washed 2× to remove unconjugated antibodies and resuspended in 60 μL of IP lysis buffer per sample. Flash-frozen pellets were thawed on ice and resuspended in 1.2 mL IP lysis buffer supplemented with 1× cOmplete Protease Inhibitor Cocktail (Roche) and 0.1 U µL−1 SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). To lyse cells, samples were sonicated using a 1/8″ sonicator probe for 1.5 min total (2 s ON, 5 s OFF) at 20% amplitude. To clear cell debris and insoluble material, lysates were centrifuged at 21,000 x g for 15 min at 4 °C, and 1 mL supernatant was transferred to a new tube. At this point, 10 µL of each sample were set aside as “input” starting material and stored at -80 °C. For immunoprecipitation, each sample was combined with 60 μL antibody-bead complex and rotated overnight at 4 °C. The next day, each sample was washed 3× with 1 mL ice-cold IP wash buffer (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM KCl, 1 mM MgCl2), using a magnetic rack to immobilize the beads in between each wash. For RIP elution, the supernatant was removed, and beads were resuspended in 750 µL TRIzol (Thermo Fisher Scientific). After 5 min incubation at RT, the supernatant containing eluted RNA was transferred to a new tube and combined with 150 μL chloroform. Samples were mixed vigorously by inversion, incubated at RT for 3 min, and centrifuged at 12,000 x g for 15 min at 4 °C. RNA was isolated from the upper aqueous phase using the RNA Clean & Concentrator-5 kit (Zymo Research) and eluted in 15 μL RNase-free water. RNA from input samples was isolated in the same manner using TRIzol and column purification. Purified RNA was stored at -80 °C before proceeding to library preparation.
For RIP-seq library preparation (input and RIP eluates), RNA was fragmented by random hydrolysis by combining 7 µL RNA, 6 µL water, and 2 µL NEBuffer 2, and heating to 92 °C for 2 min. To remove DNA and prepare RNA ends for adapter ligation, samples were treated with 2 μL TURBO DNase (Thermo Fisher Scientific) and 2 μL RppH (NEB) in the presence of 1 µL SUPERase•In RNase Inhibitor for 30 min at 37 °C. This was followed by treatment with 1 µL T4 PNK (NEB) in 1× T4 DNA ligase buffer (NEB) for 30 min at 37 °C. Reactions were column-purified using the Zymo RNA Clean & Concentrator-5 kit and eluted in 10.5 μL RNase-free water. RNA concentrations were quantified using the DeNovix RNA Assay. Illumina sequencing libraries were prepared using the NEBNext Small RNA Library Prep kit, and libraries were sequenced on an Illumina NextSeq 500 in paired-end mode with 150 cycles per end.

treatment_protocol: For experiments +/- phage infection, 40 mL cultures were split in half, and phage T5 was added to one half at a multiplicity of infection (MOI) of 5, which was calculated as the ratio of phage plaque forming units (PFU) to bacterial colony forming units (CFU). Uninfected and infected cultures were grown for 1 hr at 37 °C. For experiments without phage infection, 20 mL cultures were grown to OD600 of 0.5 and directly harvested.

tag: rip antibody

value: Monoclonal ANTI-FLAG M2 antibody (Sigma-Aldrich, cat. number:F1804)


title: Sublibary 1


title: KpnDRT2_WT_uninfected_dRNA-seq_untreated


title: KpnDRT2_WT_uninfected_dRNA-seq_treated


title: KpnDRT2_SL6_MUT_RIP


title: Retron-Eco1_RIP


title: KpnDRT2_SL1_MUT_RIP


title: KpnDRT2_YCAA_rep2_infected_RIP


title: KpnDRT2_cDNA_start_MUT_RIP


title: KpnDRT2_WT_rep1_infected_RIP


title: KpnDRT2_WT_rep3_infected_RIP


title: KpnDRT2_WT_rep2_infected_RIP


title: Section_2

description: Fresh Frozen


title: KpnDRT2_YCAA_rep2_uninfected_RIP


title: KpnDRT2_YCAA_rep3_infected_RIP


title: KpnDRT2_SL5_MUT_RIP


title: KpnDRT2_YCAA_rep1_infected_RIP


title: KpnDRT2_YCAA_rep3_uninfected_RIP


title: KpnDRT2_YCAA_rep1_uninfected_RIP


title: KpnDRT2_WT_rep1_uninfected_RIP


title: KpnDRT2_WT_rep3_uninfected_RIP


title: KpnDRT2_WT_rep2_uninfected_RIP


title: Retron-Eco1_input


title: KpnDRT2_SL6_MUT_input


title: KpnDRT2_SL2_MUT_input


title: KpnDRT2_cDNA_start_MUT_input


title: KpnDRT2_SL1_MUT_input


title: KpnDRT2_SL5_MUT_input


title: KpnDRT2_YCAA_rep1_infected_input


title: KpnDRT2_YCAA_rep3_infected_input


title: KpnDRT2_WT_rep2_infected_input


title: KpnDRT2_WT_rep3_infected_input


title: KpnDRT2_YCAA_rep2_infected_input


title: KpnDRT2_WT_rep1_infected_input


title: KpnDRT2_YCAA_rep3_uninfected_input


title: KpnDRT2_YCAA_rep2_uninfected_input


title: KpnDRT2_WT_rep3_uninfected_input


title: Retron-Eco1_cDIP

data_processing:
cDIP-seq and corresponding input datasets were processed using cutadapt (v4.2) to remove Illumina adapter sequences, trim low-quality ends from reads, and filter out reads shorter than 15 bp. Reads were mapped to combined reference files containing the MG1655 genome (NC_000913.3) and relevant plasmid sequence, as well as the T5 genome (NC_005859.1) for +/- infection experiments, using bwa-mem2 (v2.2.1) with default parameters. SAMtools (v1.17) was used to sort and index alignments. Coverage tracks were generated using bamCoverage (v3.5.1) with a bin size of 1, separation of top and bottom strand alignments, and scaling of coverage according to sequencing depth (based on the total number of reads passing initial trimming and length filtering).
Assembly: Genome reference sequences can be found in the NCBI database via the accession numbers NC_000913.3 (E. coli MG1655) and NC_005859.1 (phage T5). Plasmid reference sequences can be found in Tang et al. (2024) Table S3.
Supplementary files format and content: bigWig (.bw) files represent normalized read coverage files, separated by strand.
Library strategy: cDIP-seq

extract_protocol:
Cells were harvested by centrifugation at 4,000 x g for 10 min at 4 °C, and the supernatant was removed. The pellet was washed with 5 mL of cold TBS (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM NaCl) and spun down again as before. The supernatant was removed, and the pellet was washed with 1 mL of cold TBS before centrifugation at 10,000 x g for 5 min at 4 °C. The supernatant was removed, and the pellet was flash-frozen in liquid nitrogen and stored at -80 °C. Antibodies for immunoprecipitation were conjugated to magnetic beads as follows: for each sample, 60 μL Dynabeads Protein G (Thermo Fisher Scientific) were washed 3× in 1 mL IP lysis buffer (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM KCl, 1 mM MgCl2, 0.2% Triton X-100), resuspended in 1 mL IP lysis buffer, combined with 20 μL anti-FLAG M2 antibody (Sigma-Aldrich, F3165), and rotated for > 3 hours at 4 °C. Antibody-bead complexes were washed 2× to remove unconjugated antibodies and resuspended in 60 μL of IP lysis buffer per sample. Flash-frozen pellets were thawed on ice and resuspended in 1.2 mL IP lysis buffer supplemented with 1× cOmplete Protease Inhibitor Cocktail (Roche) and 0.1 U µL−1 SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). To lyse cells, samples were sonicated using a 1/8″ sonicator probe for 1.5 min total (2 s ON, 5 s OFF) at 20% amplitude. To clear cell debris and insoluble material, lysates were centrifuged at 21,000 x g for 15 min at 4 °C, and 1 mL supernatant was transferred to a new tube. At this point, 10 µL of each sample were set aside as “input” starting material and stored at -80 °C. For immunoprecipitation, each sample was combined with 60 μL antibody-bead complex and rotated overnight at 4 °C. The next day, each sample was washed 3× with 1 mL ice-cold IP wash buffer (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM KCl, 1 mM MgCl2), using a magnetic rack to immobilize the beads in between each wash. For cDIP elution, the supernatant was removed, and beads were resuspended in 90 µL IP wash buffer and treated with 5 µg RNase A (Thermo Fisher Scientific) for 30 min at 37 °C. Input samples were adjusted to 90 µL with IP wash buffer and treated with RNase A in parallel. SDS was added to IP and input samples to a final concentration of 1%, and samples were treated with 25 µg Proteinase K (Thermo Fisher Scientific) for 30 min at 55 °C. Beads were immobilized using a magnetic rack, and the supernatant containing eluted DNA was transferred to a new tube. DNA was isolated using the Monarch PCR and DNA Cleanup kit (NEB), following the Oligonucleotide Cleanup protocol and eluting in 15 µL DNase-free water. For Retron-Eco1 samples, DNA was treated with DBR1 (Origene) in reactions containing 2 µL DNA, 0.5 µL DBR1, 1× rCutSmart in 10 µL total volume, in order to cleave the 2′-5′ phosphodiester linkage between msRNA and msDNA. Reactions were cleaned up using the Monarch PCR and DNA Cleanup kit (NEB), with elution in 15 µL DNase-free water. Purified DNA was stored at -80 °C before proceeding to library preparation.
For cDIP-seq library preparation, 2 µL of each input sample and 10 µL of each IP eluate were diluted to 15 µL with DNase-free water. Samples were denatured by heating at 95 °C for 2 min, and then immediately placed on ice. Ligation of Illumina adapters and conversion of ssDNA to dsDNA were performed using the xGen ssDNA & Low-Input DNA Library Prep Kit (IDT), and libraries were sequenced on an Illumina NextSeq 500 in paired-end mode with 150 cycles per end.

tag: cdip antibody


title: KpnDRT2_cDNA_start_MUT_cDIP

tag: strain


title: KpnDRT2_SL5_MUT_cDIP


title: KpnDRT2_WT_rep2_uninfected_input


title: KpnDRT2_SL6_MUT_cDIP


title: KpnDRT2_YCAA_rep1_uninfected_input


title: KpnDRT2_YCAA_rep3_infected_cDIP


title: KpnDRT2_SL1_MUT_cDIP


title: KpnDRT2_WT_rep1_uninfected_input


title: KpnDRT2_YCAA_rep1_infected_cDIP


title: KpnDRT2_YCAA_rep2_infected_cDIP


title: KpnDRT2_SL2_MUT_cDIP


title: KpnDRT2_WT_rep1_infected_cDIP


title: KpnDRT2_WT_rep3_infected_cDIP


title: KpnDRT2_WT_rep2_infected_cDIP


title: KpnDRT2_YCAA_rep2_uninfected_cDIP


title: KpnDRT2_YCAA_rep3_uninfected_cDIP


title: KpnDRT2_WT_rep3_uninfected_cDIP


title: KpnDRT2_YCAA_rep1_uninfected_cDIP


title: KpnDRT2_WT_rep2_uninfected_cDIP


title: KpnDRT2_WT_rep1_uninfected_cDIP


title: KpnDRT2_SL6_MUT_input


title: KpnDRT2_SL5_MUT_input


title: KpnDRT2_SL2_MUT_input


title: KpnDRT2_YCAA_rep3_infected_input


title: KpnDRT2_YCAA_rep2_infected_input


title: KpnDRT2_YCAA_rep1_infected_input


title: KpnDRT2_WT_rep3_infected_input


title: KpnDRT2_WT_rep1_uninfected_input


title: KpnDRT2_WT_rep2_infected_input


title: KpnDRT2_WT_rep2_uninfected_input


title: KpnDRT2_YCAA_rep2_uninfected_input


title: KpnDRT2_WT_rep3_uninfected_input


title: KpnDRT2_YCAA_rep3_uninfected_input


title: KpnDRT2_YCAA_rep1_uninfected_input


title: KpnDRT2_WT_rep1_infected_input


title: Murine Colon CD45+ Run 1

status: Public on Jul 22 2024

city: Regensburg

first: LIT

last: NGS-Core

address: Franz-Josef-Strauß-Allee 11

department: NGS-Core

country: Germany

institute: Leibniz Institute for Immunotherapy

data_processing:
Fragment files were used to create Arrow files using the package ArchR (v.1.0.2) in R (v.4.0.0) and barcodes with >3000 fragments and a TSS enrichment score > 6 were maintained. Additionally, barcodes were excluded if they were flagged as gel bead multiplet, low-targeting barcode or barcode multiplet by Cell Ranger atac.
Potential cell doublets were identified with ArchR and barcodes with a DoubletScore >=2 were filtered out.
Dimensionality reduction with an iterative LSI  approach and UMAP and graph-based clustering were done with ArchR.
MACS2 (v.2.2.6) was used for peak calling at a cluster resolution of 0.1.
Gene activity scores were calculated with ArchR and transcription factor deviation scores were added using chromVAR (v.1.10.0). Fragments overlapping transposable elements were quantified based on integration sites from the RepeatMasker annotation available at http://hgdownload.soe.ucsc.edu/goldenPath/mm10/database/rmsk.txt.gz
Assembly: mm10
Supplementary files format and content: Output from Cell Ranger atac is described at https://support.10xgenomics.com/single-cell-atac/software/pipelines/latest/what-is-cell-ranger-atac.
Supplementary files format and content: More information on the structure of Arrow files and ArchR projects is available at https://www.archrproject.com/bookdown/index.html

source_name: colon

growth_protocol: SPF mice were housed under specified pathogen-free conditions at the central animal facility of the university clinics in Regensburg.

extract_protocol: Sorted cells were pelleted by centrifugation (300xg, 5min 4°C). Supernatant was removed and cells were resuspended in 50 µl - 100 µl 0.04%BSA-PBS buffer. Cells were centrifuged (300xg, 5 min 4°C), supernatant was completely removed and chilled nuclei lysis buffer (10 mM TRIS-HCL pH7.4, 10mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40, 0.01% Digitonin, 1% BSA in nuclease-free water) was added. Lysis was performed for 2 min at 4°C, followed by addition of 50 µl washing buffer (10 mM TRIS-HCl ph7.4, 10 mM NaCl, 3 mM MgCL2, 1%BSA, 0.1% Tween-20 in nuclease-free water). After centrifugation (300xg, 5min 4°C), supernatant was removed and 45 µl chilled diluted nuclei buffer (10X Genomics) was added. After centrifugation (300xg, 5 min 4°C), supernatant was removed and 7µl chilled diluted nuclei buffer (10X Genomics) was added. Nuclei were counted (where applicable) and transposition mix was prepared with 5 µl of nuclei suspension based on the manufacturer’s protocol. Transposition was performed for one hour at 37°C, followed by supplementation of master mix and beads (Single Cell ATAC Gel Beads V1.0 or V1.1 and reagents, 10X Genomics #1000175), loading on 10X Chromium Next GEM Chip H (10X Genomics #1000161) and processing on 10X Chromium Controller (10X Genomics #120212). GEM incubation was performed with 11 cycles (mouse CD45) 10-11 cycles (human CD45) of PCR. All samples were processed in duplicate runs to increase total output of cells for analysis. Library was prepared according to the protocol with cycle numbers dependent on input nuclei concentration. scATAC libraries were sequenced on an Illumina NextSeqTM 550 with NextSeqTM 500/550 High Output Kit v2.5 (75 cycles, Illumina #20024906).

treatment_protocol: T cells were isolated and pre-enriched as described in the associated publication. Afterwards, they were stained with live/dead dye and surface antibodies as described. Cells were then sorted on an BDTM FACSAriaTM IIu or FACSAriaTM FUSION cell sorter using 85 µm nozzle size and 4-way-purity sort mode into 1.5 ml tubes filled with FACS buffer. Sample and post-sort quality control were recorded where applicable and are displayed in supplementary figures of the manuscript.

value: colon

tag: cell type

value: CD45+ cells

value: Run 1


title: Murine Colon CD45+ Run 2

tag: cell line

value: Run 2


title: Murine VAT CD45+ Run 1

source_name: vat

value: vat

tag: treatment


title: Murine VAT CD45+ Run 2

tag: genotype


title: N2_sRNA-seq_rep3

status: Public on Jul 17 2024

city: Bloomington

first: Leanne

last: Kelley

state: IN

address: 1001 E Third St

institute: Indiana University

data_processing:
Read were trimmed and quality-controlled using cutadapt with the parameters ‘-a AGATCGGAAGAGCACACGTCTGAACTCCAGTC -q 20 -m 15 -M 26’. Mapping was performed with HISAT2 using the c_elegans.PRJNA13758.WS284.genomic.fa reference genome and sorted BAM files were used as input into smalldisco. miRNAs and piRNAs were extracted from c_elegans.PRJNA13758.WS272.canonical_geneset.gtf and the file was converted into a BED file using BEDOP’s gft2bed command. The sorted BAM files, BED reference, and c_elegans.PRJNA13758.WS284.genomic.fa reference were used as input for smalldisco’s tail function using the ‘--tails-all’ to map the reads as sense to strand for miRNAs and piRNAs. For siRNAs, the sorted BAM files and c_elegans.PRJNA13758.WS272.canonical_geneset.gtf reference were supplied for the sirna function (smalldisco) with the parameter ‘-f CDS’. The output of this was used as input with smalldisco’s tail function, along with the c_elegans.PRJNA13758.WS284.genomic.fa reference. Counts tables were imported into R and processed to produce a matrix that combines the miRNA, piRNA, and siRNA counts (regardless of tail type).
Assembly: C. elegans WS284
Supplementary files format and content: Total counts (not parsed by tail type or sRNA biotype) in a matrix

organism: Caenorhabditis elegans

source_name: whole animal

growth_protocol: 1-day old adult hermaphrodites raised at 22°C

extract_protocol:
RNA was isolated from whole animals using the TRIzol (Invitrogen) method.
Treated with RNA 5' polyphosphatase; libraries generated with NEBNext Small Library Prep Kit.

value: whole animal

tag: developmental stage

value: 1-day adult

tag: gender

value: hermaphrodite


title: Murine Skin CD45+ Run 2

source_name: skin

value: skin


title: N2_sRNA-seq_rep2


title: Murine Skin CD45+ Run 1


title: N2_sRNA-seq_rep6


title: N2_sRNA-seq_rep5


title: N2_sRNA-seq_rep1


title: N2_sRNA-seq_rep4


title: pup-3(tm5089);pup-4(om141);pup-1/-2(om129)_sRNA-seq_rep3


title: Retron-Eco1_input


title: pup-3(tm5089);pup-4(om141);pup-1/-2(om129)_sRNA-seq_rep2


title: KpnDRT2_cDNA_start_MUT_input


title: pup-3(tm5089);pup-4(om141);pup-1/-2(om129)_sRNA-seq_rep1


title: KpnDRT2_SL1_MUT_input


title: pup-3(tm5089);pup-1/-2(om129)_sRNA-seq_rep2


title: pup-3(tm5089);pup-1/-2(om129)_sRNA-seq_rep1


title: pup-1/-2(om129)_sRNA-seq_rep3


title: pup-1/-2(om129)_sRNA-seq_rep2


title: pup-3(tm5089)_sRNA-seq_rep2


title: pup-4(om141)_sRNA-seq_rep1


title: pup-3(tm5089)_sRNA-seq_rep1


title: pup-1/-2(om129)_sRNA-seq_rep1


title: pup-4(om141)_sRNA-seq_rep3


title: pup-4(om141)_sRNA-seq_rep2


title: pup-1(tm1021)_sRNA-seq_rep3


title: pup-2(tm4344)_sRNA-seq_rep1


title: N2_mRNA-seq_rep4

data_processing:
Basecalled with guppy_basecaller (v4.4.2), demultiplexed with guppy_barcoder (v4.4.2), FASTQ chunks within each genotype/replicate were combined into one FASTQ file using the cat command in Unix, reads were mapped to the transcriptome using minimap2 with the parameters '-ax map-ont --MD -k14' and using the RefSeq GCF 000002985.6 reference. In parallel, the non-basecalled FAST5 files were demultiplexed with the demux_fast5 (v4.1.1) command from the ont_fast5_api software and these were used as input for the find_tails command in the program tailfindr (polyu branch). The resulting tails count table and the SAM file from the mapping step were used as input for the annotate_tails function in tailfinder to produce the final counts table. This was loaded into R to convert to a matrix.
Assembly: RefSeq GCF 000002985.6
Supplementary files format and content: Total counts (not parsed by tail type) in a matrix

growth_protocol: 1 day old adult hermaphrodites raised at 22°C

extract_protocol:
RNA was isolated from whole animals using the TRIzol (Invitrogen) method.
Ribosomal-depleted RNA was subjected to the Nano3P-seq protocol (modification of the ONT direct cDNA-seq kit, published by Begik et al. 2023).


title: pup-1(tm1021)_sRNA-seq_rep2


title: pup-2(tm4344)_sRNA-seq_rep3


title: N2_mRNA-seq_rep5


title: pup-1(tm1021)_sRNA-seq_rep1


title: pup-2(tm4344)_sRNA-seq_rep2


title: pup-3(tm5089)_sRNA-seq_rep3


title: pup-1/-2(om129)_mRNA-seq_rep2


title: pup-1/-2(om129)_mRNA-seq_rep3


title: N2_mRNA-seq_rep3


title: N2_mRNA-seq_rep1


title: pup-4(om141)_mRNA-seq_rep1


title: pup-3(tm5089);pup-1/-2(om129)_sRNA-seq_rep3