Rna isolation kit

The hTBM were lysed by agitating for 2 min at 50 rpm in lysis buffer containing β-Mercaptoethanol prior to RNA isolation at 4°C. Total RNA was isolated using the Qiagen RNA isolation kit (Qiagen, Germany) according to the manufacturer’s instruction. Isolated total-RNA quality and quantity was determined using TECAN infinite M200 microplate reader (TECAN GmbH, Germany). 1ug of the total RNA was transcribed to cDNA with iScript™ cDNA synthesis kit (Bio-Rad, USA). The cDNA was quantified with the SSOFast™ EvaGreen^® Supermix (Bio-Rad, USA) on the Sens Quest CFX96 real time PCR thermocycler (Bio-Rad, USA). The GAPDH gene was used as reference in all the quantitative RT-PCR experiments. Primers were designed using Primer3 (Supplementary Table 2) and were used at a final concentration of 500nM. A 3-cycle amplification method was performed with cycling conditions as follows: (i) initial denaturation at 95°C 3 min, followed by 40 cycles of (ii) denaturation at 95°C for 10 s, (iii) primer annealing at 65°C for 30 s and (iv) stabilization, elongation and fluorescence detection at 72°C for 30 s. The gene expression fold change was calculated using the 2^(-ΔΔCT) Livak threshold cycle (C_T) method (Livak & Schmittgen, 2001 (link))

Total RNA samples were extracted from NPCs, neurons, and astrocytes using a Qiagen RNA Isolation Kit (Qiagen, Hilden, Germany) with DNAase I treatment (Qiagen). mRNA was isolated from total RNA using a Dynabeads mRNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA). mRNA samples (200 ng) were used to construct RNA libraries using MGIEasy RNA Library Prep kit (MGI, Shenzhen, China) and sequenced with MGISEQ-2000 sequencing platform. We used Salmon software [89 (link)] with default parameters to map RNA-seq reads to the human transcriptome release 37 from GENCODE (Frankish et al., 2019). The same software was used to count the number of RNA reads mapped to each reference transcript. The transcript-level quantification results were converted to the gene-level quantities with tximport [90 (link)] R package. Finally, DESeq2 [91 (link)] R package was used to normalize the gene-level quantities and plot PCA and heatmap from the normalization results.

RNA isolation was performed using Qiagen RNA isolation kit (#74106, Qiagen, Hilden, Germany) as per the product manual. One microgram of RNA was used to synthesize cDNA using ProtoScript® II Reverse Transcriptase, NEB (#M0368L, New England Biolabs (NEB), Ipswich, USA). Real-time PCR was performed by mixing 10 ng of cDNA, 0.5 µM of forward and reverse primer each (final concentration; Supplementary Table S2) and SyBr green dye (#204057, Qiagen, Hilden, Germany). A LightCycler 480 was used to perform the RT-PCR.

Total RNA was isolated from tumor tissues of patients with osteosarcoma using Qiagen RNA isolation kit (Qiagen). cDNA was synthesized using SuperScript II reverse transcriptase with random hexamers (Life Technologies). To quantitate the expression of human CD47 mRNA, real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Melting curve analysis was done at the end of the reaction to assess the quality of the final PCR products. The threshold cycle C(t) values were calculated by fixing the basal fluorescence at 0.05 units. Three replicates were used for each sample, and the average C(t) value was calculated. The ΔC(t) values were calculated as C(t) sample - C(t) GAPDH. The N-fold increase or decrease in expression was calculated by the ΔΔCt method using the C(t)GAPDH value as the reference point.

RNA was extracted from ES‐derived Day 17 erythroblasts which are orthochromatic erythroblasts using QIAGEN RNA isolation kit (QIAGEN#74104) according to the manufacturer's instructions. The RNA integrity number of each sample was >9. Approximately 100 ng of total RNA was used for construction of cDNA library by Illumina TruSeq kit (Illumina #RS‐121‐2001, #RS‐121‐2002). Sequencing was performed on Illumina HiSeq 4000 with 150 bp paired‐end. RNA‐Seq data of CB‐derived erythroblasts were downloaded from our previous data.³³ Raw bulk RNA‐seq data of ES‐ortho were filtered by fastp to remove low quality reads. Reads were mapped to UCSC hg19 by STAR³⁴ and quantified by FeatureCounts.³⁵ Gene expression was analysed using DESeq2.³⁶ Principal component analysis (PCA) was performed on log‐transformed normalized counts for expressed genes. Differentially expressed genes were identified as fold change ≥4 and adjusted p‐value ≤0.05. GO (Gene ontology) enrichment analysis were performed by Metascape.³⁷ GO term was considered as significant with q‐value <0.001. RNA‐seq data are available at GEO under accession number GSE179778. Time series analysis was applied by R package TCseq.