RNA was isolated from tumors or tumor cells using RNeasy columns (Qiagen). For TAM sequencing, macrophages were isolated by FACS using the antibody panel described above, and RNA was isolated using RNeasy columns (Qiagen). RNA was sequenced using the Illumina HiSeq 4000 libraries and sequencing platform with 50 base pair single end reads by the Duke GCB Sequencing and Genomic Technologies Shared Resource (Durham, NC). Sequencing data have been deposited in SRA as PRJNA506006 for cell line data and PRJNA505845 for macrophage data.
Rneasy column
Manufactured by Qiagen
Sourced in United States, Germany, United Kingdom, Canada, Spain, Netherlands, Australia, Japan, Italy
The RNeasy columns are lab equipment used for the purification and isolation of RNA from various biological samples. They provide a reliable and efficient method for extracting high-quality RNA, which is essential for a wide range of molecular biology and research applications.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (170 mentions)
Trizol reagent, by Thermo Fisher Scientific (149 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (62 mentions)
Iscript cdna synthesis kit, by Bio-Rad (28 mentions)
2100 bioanalyzer, by Agilent Technologies (27 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (24 mentions)
Rneasy kit, by Qiagen (17 mentions)
Bioanalyzer, by Agilent Technologies (17 mentions)
Hiseq 2000, by Illumina (16 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (15 mentions)
Nextseq 500, by Illumina (14 mentions)
Low input quick amp labeling kit, by Agilent Technologies (14 mentions)
Hiseq 2500, by Illumina (14 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (14 mentions)
Tri reagent, by Merck Group (13 mentions)
Nanodrop, by Thermo Fisher Scientific (13 mentions)
Sybr green master mix, by Thermo Fisher Scientific (13 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (12 mentions)
Taqman probe, by Thermo Fisher Scientific (12 mentions)
885 protocols using rneasy column
1
RNA Isolation and Sequencing from Tumors
2
Extracting RNA from Hydrothermal Chimneys
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A chimney structure was obtained from the Noho site of the Sakai field (27°31.386′N, 126°59.209′E) (34 ), Mid-Okinawa Trough, Japan, at a depth of 1,550 m by means of the ROV Hyper-Dolphin (Dive#1860) during R/V Natsushima cruise NT15-13 (JAMSTEC) in August 2015. Immediately after its recovery onboard, the chimney structure was stored at −80°C until used.
The chimney sample was pulverized with a mortar and pestle to a fine powder of micron-size particles in liquid nitrogen. Total RNA was directly extracted from 1.79–1.92 g of the sample using TRIzol reagent (Thermo Fisher Scientific) and the RNeasy column (Qiagen), in the presence or absence of 100 μL of 0.6 M STPP. In addition, the mixture of the pulverized chimney structure and STPP was sonicated using TAITEC VP-050 (TAITEC, Koshigaya, Japan) at 10W for 20 s. After 30 min on ice, the supernatant was recovered, and RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) and the RNeasy column (Qiagen) (indirect extraction). STPP was used in the indirect RNA extraction procedure because the supernatant potentially includes tiny particles of the chimney structure and/or RNA accidentally released from cells by sonication. RNA quality and the 16S rRNA copy number were evaluated as described above.
The chimney sample was pulverized with a mortar and pestle to a fine powder of micron-size particles in liquid nitrogen. Total RNA was directly extracted from 1.79–1.92 g of the sample using TRIzol reagent (Thermo Fisher Scientific) and the RNeasy column (Qiagen), in the presence or absence of 100 μL of 0.6 M STPP. In addition, the mixture of the pulverized chimney structure and STPP was sonicated using TAITEC VP-050 (TAITEC, Koshigaya, Japan) at 10W for 20 s. After 30 min on ice, the supernatant was recovered, and RNA was extracted using TRIzol reagent (Thermo Fisher Scientific) and the RNeasy column (Qiagen) (indirect extraction). STPP was used in the indirect RNA extraction procedure because the supernatant potentially includes tiny particles of the chimney structure and/or RNA accidentally released from cells by sonication. RNA quality and the 16S rRNA copy number were evaluated as described above.
3
Zebrafish Retina RNA Isolation and qRT-PCR
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Dorsal or whole retinas were isolated from light-damaged albino zebrafish (0, 36, 48, 60, 72, 84, 96 hLT and 2, 5, and 7 drec) or metronidazole-treated albino;Tg[rho:Eco.NfsB-EGFP]nt19 zebrafish (0, 24, 48, 72, 96, and 120 h after metronidazole treatment onset), respectively. Retinas were homogenized in TRIzol following the manufacturer’s instructions to the step of phase separation, when the clear phase was mixed with 75% ethanol before transferring the mixture onto a RNeasy column (Qiagen) The RNeasy kit protocol was followed starting at the step of the RNeasy column and included a 15 min DNase step (Qiagen) according to manufacturer’s instructions. The resultant RNA was stored at −80°C. cDNA was prepared using qScript cDNA super mix (QuantaBio) and subjected to quantitative real-time polymerase chain reaction (qRT-PCR) as previously described (Gorsuch et al., 2017 (link)) using primers listed in Table 2 .
4
Total RNA Extraction and Sequencing
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Total RNA was extracted from T cells using Qiazol (Qiagen, IT), purified from DNA contamination through a DNase I (Qiagen, IT) digestion step and further enriched by Qiagen RNeasy columns (Qiagen, IT). Integrity of RNA was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, CA). RNA libraries for sequencing were generated in triplicate using 500 ng of RNA for each sample according to the Illumina TruSeq Stranded Total RNA kit with an initial ribosomal depletion step by using Ribo Zero Gold (Illumina, CA). Libraries were sequenced in a paired-end mode (2 × 75 bp) with NextSeq 500 (Illumina, CA). For each sample generated by the Illumina platform, a pre-process step for quality control was performed to assess sequence data quality and to discard low-quality reads.
5
RNA Extraction and cDNA Synthesis Protocol
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According to the manufacturer’s instructions, approximately 107 cells
were lysed in 1 ml of TRIzol reagent (Invitrogen). DNase treatment (Qiagen,
Hamburg, Germany) was performed, and total RNA was purified with Qiagen RNeasy
Columns (Qiagen). With random hexamer primers, cDNAs were synthesized from 1 μg
of total RNA using reverse transcriptase (Applied Biosystems Inc., Foster City,
CA, USA) and real-time polymerase chain reaction (RT-PCR) with SYBR Green
(Applied Biosystems Inc.).
were lysed in 1 ml of TRIzol reagent (Invitrogen). DNase treatment (Qiagen,
Hamburg, Germany) was performed, and total RNA was purified with Qiagen RNeasy
Columns (Qiagen). With random hexamer primers, cDNAs were synthesized from 1 μg
of total RNA using reverse transcriptase (Applied Biosystems Inc., Foster City,
CA, USA) and real-time polymerase chain reaction (RT-PCR) with SYBR Green
(Applied Biosystems Inc.).
6
Microarray Analysis of T Cell Subsets
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Affymetrix microarrays were performed as described [42 ]. Total RNA was extracted from thymic CD4-CD8+CD3lo/- and CD4-CD8+CD3+ T cells with Trizol and purified over Qiagen RNeasy columns (Qiagen). Synthesis and labeling of RNA and hybridization of arrays was conducted. Stained arrays (430 2.0) were scanned on an Agilent Gene Array Scanner (Affymetrix). Most significantly changed mRNA transcripts are shown.
7
Quantification of hCAMP Expression in Macrophages
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Infected macrophages (untreated, GM-CSF treated, or Vitamin D treated) were lysed in Trizol for trizol-chloroform preparation of RNA. RNA was precipitated using isopropanol and then processed using Qiagen RNEasy columns (Qiagen). RNA was reverse transcribed into cDNA using SuperScript III (ThermoFisher Scientific) and quantitative PCR for hCAMP and GAPDH was done using iTAQ Universal Sybr Green (Bio-Rad). The primer sequences for GAPDH are: F: 5’-GTCTCCTCTGACTTCAACAGCG-3’ and R: 5’-ACCACCCTGTTGCTGTAGCCAA-3’. The primer sequences for hCAMP are: F: 5’-GGACCCAGACACGCCAAA-3’, R: 5’-GCACACTGTCTCCTTCACTGTGA-3’.
8
RNA Extraction from Irradiated PBMC and Whole Blood
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RNA from irradiated and sham-irradiated PBMC samples was extracted 8 h after irradiation for microarray hybridisation and 8 and 24 h for qRT-PCR validation. RNA from whole blood samples used for qRT-PCR validation was extracted 8 and 24 h after irradiation. For RNA isolation from PBMCs, a combined approach consisting of the TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) extraction method and purification on Qiagen RNeasy columns (Qiagen, Venlo, The Netherlands), was used. More detailed information on the RNA extraction procedure can be found in the Supplementary Methods . The QIAamp RNA Blood Mini Kit (Qiagen, Venlo, The Netherlands) was used to extract RNA from whole blood samples. The starting quantity of blood was 1.5 ml per sample. All procedures were performed following the manufacturer’s instructions. RNA concentration was measured on a NanoDrop-2000 spectrophotometer (Thermo Scientific, Erembodegem, Belgium) and the quality of total RNA samples was assessed using Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA, USA). All samples had a RNA Integrity Number >8 and were therefore considered as suitable for further processing for microarrays and qRT-PCR.
9
RNA Extraction and Microarray Hybridization
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Total RNA was independently extracted from each of the 48 cell culture wells using QIAshredder homogenates and QIAgen RNeasy columns (Qiagen), incorporating the on-column DNase treatment (Ambion) to remove genomic DNA contamination. RNA integrity and purity was assayed visually by gel electrophoresis and additionally by A260/280 spectrophotometry. All the RNA samples resolved into discrete 16S and 26S ribosomal RNA bands.
Because each of the four treatment groups comprised 12 independent RNA samples, these 12 were pooled into 3 major groups of 4 replicates with each replicate contributing 0.5 μg. This process yielded 12 by 2.0 μg pooled RNA samples as follows (no transfection 1, no transfection 2, no transfection 3, empty construct 1, empty construct 2, empty construct 3, short transcript 1, short transcript 2, short transcript 3, long transcript 1, long transcript 2, long transcript 3).
The 12 RNA pools were submitted to the Gene Expression Centre at the University of Queensland’s Institute for Molecular Bioscience for cDNA synthesis and hybridisation to the Illumina WG6 mouse microarray platform. This facility performed an additional set of quality checks based on possession of an RNA Integrity Number (RIN) >8 assayed on a Bioanalyzer.
Because each of the four treatment groups comprised 12 independent RNA samples, these 12 were pooled into 3 major groups of 4 replicates with each replicate contributing 0.5 μg. This process yielded 12 by 2.0 μg pooled RNA samples as follows (no transfection 1, no transfection 2, no transfection 3, empty construct 1, empty construct 2, empty construct 3, short transcript 1, short transcript 2, short transcript 3, long transcript 1, long transcript 2, long transcript 3).
The 12 RNA pools were submitted to the Gene Expression Centre at the University of Queensland’s Institute for Molecular Bioscience for cDNA synthesis and hybridisation to the Illumina WG6 mouse microarray platform. This facility performed an additional set of quality checks based on possession of an RNA Integrity Number (RIN) >8 assayed on a Bioanalyzer.
10
Isolation and Characterization of Nucleic Acids from Mononuclear Cells
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