CD127 imprints functional heterogeneity to diversify monocyte responses in human inflammatory diseases

Article activity feed

1. 

   ScreenIT

   Nov 11, 2020

   SciScore for 10.1101/2020.11.10.376277: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement

   IRB: Cell culture and reagents: PBMCs of anonymous healthy donors were isolated from buffy coats purchased from the Beijing Red Cross Blood Center using density gradient cell separation by Ficoll (Lymphoprep™, STEMCELL Technologies) following the protocol approved by the Institutional Review Board of School of Medicine, Tsinghua University. IACUC: Mice: The laboratory animal facility at Tsinghua University has been accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International), and the IACUC (Institutional Animal Care and Use Committee) of Tsinghua University approved the protocol used in this study for blood …

   SciScore for 10.1101/2020.11.10.376277: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Institutional Review Board Statement	IRB: Cell culture and reagents: PBMCs of anonymous healthy donors were isolated from buffy coats purchased from the Beijing Red Cross Blood Center using density gradient cell separation by Ficoll (Lymphoprep™, STEMCELL Technologies) following the protocol approved by the Institutional Review Board of School of Medicine, Tsinghua University. IACUC: Mice: The laboratory animal facility at Tsinghua University has been accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International), and the IACUC (Institutional Animal Care and Use Committee) of Tsinghua University approved the protocol used in this study for blood collection from mouse cheeks.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Lung tissue sections were stained with hematoxylin for assessment of pulmonary architecture, and anti-CD68 (ab201340, abcam) and anti-CD127 (PA5-97870, Invitrogen) antibodies were used for immunohistochemistry.	anti-CD68 suggested: (Antibodies.com Cat# A86316, RRID:AB_2747830) anti-CD127 ( PA5-97870 , Invitrogen ) suggested: None
   The tissues were then incubated with indicated primary antibodies diluted (anti-CD68, 1:100; anti-CD127, 1:500) in PBS/5% goat serum at 4°C overnight, and washed in PBS/0.2% Tween-20 at room temperature for 30 min three times.	anti-CD127 suggested: (Thermo Fisher Scientific Cat# MA1-82689, RRID:AB_927531)
   The tissues were incubated with Alexa dye-conjugated secondary antibodies (Alexa Fluor™ 488 goat anti-mouse IgG, 1:500, B40941, Life Technologies; Alexa Fluor™ 555 goat anti-rabbit IgG, 1:500, A27039, Invitrogen) and DAPI (1:200, C0060-1, Solarbio) in PBS/0.5% BSA at room temperature for 2 h and washed in PBS/0.2% Tween-20 at room temperature for 1 h five times before mounting with SlowFade Diamond Antifade Mountant (S36963, Life Technologies).	anti-mouse IgG suggested: None B40941 suggested: None anti-rabbit IgG suggested: (Thermo Fisher Scientific Cat# A27039, RRID:AB_2536100) A27039 suggested: (Thermo Fisher Scientific Cat# A27039, RRID:AB_2536100) S36963 suggested: None
   The DNA-protein complexes were immunoprecipitated using 5.0 μl per sample of STAT5 antibody (9363S, Cell Signaling Technology), and IgG (2729P, Cell Signaling Technology) control was performed in an equally allocated DNA-protein complexes fraction as STAT5 ChIP samples.	STAT5 suggested: (Cell Signaling Technology Cat# 9363, RRID:AB_2196923) 9363S , Cell Signaling Technology) , and IgG ( 2729P , Cell Signaling Technology ) suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Chromatin immunoprecipitation (ChIP) assay: For STAT5 ChIP assays, THP-1 cells were stimulated with LPS (100 ng/ml) for 6 h and subsequently with IL-7 (10 ng/ml) for 30 min.	THP-1 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   C57BL/6J mice were bred and housed in isolated ventilated cages (maxima six mice per cage) at the specific pathogen free facility at Tsinghua University.	C57BL/6J suggested: RRID:IMSR_JAX:000664)
   Software and Algorithms
   Sentences	Resources
   After staining, cells were washed three times with staining buffer and re-suspended in PBS for analysis in BD FACSFortessa or for fluorescence-activated cell sorting (FACS) in BD FACSAria III.	BD FACSFortessa suggested: None
   Further data analysis was implemented using Flowjo software (Tree star).	Flowjo suggested: (FlowJo, RRID:SCR_008520)
   Relative density of blotting bands was quantified using Image J (v1.52a)	Image J suggested: (ImageJ, RRID:SCR_003070)
   Non-targeting siRNA from GenePharma was used as control.	GenePharma suggested: None
   Pair-end ATAC-seq reads were aligned to human genome (UCSC hg38) using Bowtie2 (v2.2.5)20 to generate alignment files of uniquely mapped pair-end fragments with maximum length in 1000 bp and no more than one mismatch for each alignment seed with 15 bp in length.	Bowtie2 suggested: (Bowtie 2, RRID:SCR_016368)
   ChIP-seq reads in fastq files were aligned to human genome (UCSC hg38) using Bowtie (v1.1.2)21 to generate alignment files of uniquely mapped reads with maximum allowed mismatch of 2 (-m 1 -n 2) for each alignment seed.	Bowtie suggested: (Bowtie, RRID:SCR_005476)
   RNA-seq data were collected and single-end reads were aligned to human genome hg38 using TopHat (v2.1.0)22 with the parameters --min-segment-intron 50 --no-novel-indels --no-coverage-search, and only uniquely mapped reads were preserved.	TopHat suggested: (TopHat, RRID:SCR_013035)
   To identify the differentially opened chromatin regions between CD127high and CD127low monocytes, the ATAC-seq fragments were counted in each OCR for each sample by FeatureCounts (v1.5.0)23.	FeatureCounts suggested: (featureCounts, RRID:SCR_012919)
   The normalized fragments count was used to identify differentially opened chromatin regions by edgeR (v3.28.1)24.	edgeR suggested: (edgeR, RRID:SCR_012802)
   To visualize ATAC-seq and ChIP-seq signals around open chromatin regions of interest, we firstly counted ATAC-seq fragments (-fragLength given) and ChIP-seq extended reads (-fragLength 150) every 10 bp from the center of OCR to ± 2.5 kb regions for each OCR by using annotatePeaks.pl program in HOMER (v4.7.2)25.	ChIP-seq suggested: (ChIP-seq, RRID:SCR_001237) HOMER suggested: (HOMER, RRID:SCR_010881)
   RNA-seq data analyses: For coverage of mapped RNA-seq reads in transcripts, the expression level of each gene transcript was calculated as normalized reads count per kilobase of transcript per million mapped reads (FPKM) using Cufflinks (v2.2.1)26.	Cufflinks suggested: (Cufflinks, RRID:SCR_014597)
   Differential gene expression between CD127high and CD127lwo monocytes from three donors was identified using DESeq2 (v1.27.9)27.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Specifically, splicing-aware aligner STAR was used in FASTQs alignment.	STAR suggested: (STAR, RRID:SCR_015899)
   Statistical analyses were performed using GraphPad Prism Software (GraphPad Software Inc.,	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   Results from OddPub: Thank you for sharing your data.

   ---

   Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

   ---

   Results from TrialIdentifier: No clinical trial numbers were referenced.

   ---

   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

   ---

   Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 16, 18 and 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

   ---

   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

   ---

   Read the original source
2. 

   Version published to 10.1101/2020.11.10.376277 on bioRxiv

   Nov 10, 2020