GSM7611257: CD34_Diff_Day4_PROseq; Homo sapiens; OTHER
CD34+ cells were purified from human cord blood (Cord Blood Bank of Beijing) using the MACS MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The CUT&Tag assay was performed as previously described48 and followed the manufacturer's instructions for the kit (Vazyme, Nanjing, China). Briefly, 100,000 cells were freshly collected and captured using Concanavalin A (ConA) beads at room temperature. The cell/ConA beads complex was permeabilized and incubated with the primary antibody (at 1:100 dilution) overnight at 4 °C. The secondary antibody was added to the solution and incubated at room temperature for 1 h. The secondary antibody was washed off prior to adding pA/G-Tn5 to the cell suspension and then incubated at room temperature for 1 h. The adapters were then inserted into the Tn5-tagged genome using the TruPrep Tagment Buffer L (TTBL). The genomic DNA was extracted with DNA beads, and the tagged DNA fragments were amplified using the Next-Generation Sequencing (NGS) adapters with 16 cycles of PCR. The adaptor dimer was removed from the PCR products with DNA Clean Beads (Vazyme, Nanjing, China). The library was quantified before sequencing. 20 millions Day 4 or Day 16 ex vivo-cultured permeabilized CD34 cell nuclei were added to a 2 × Nuclear Run-On (NRO) reaction mixture and incubated for 3 min at 37 °C. Nascent RNA was extracted and fragmented by base hydrolysis in 0.2 N NaOH on ice for 10 min and neutralized by adding a 1× volume of 1 M Tris-HCl pH 6.8. Fragmented nascent RNA was subjected to buffer exchange and purified using streptavidin beads and ligated with reverse 3' RNA adapter VRA3 (5'p-GAUCGUCGGACUGUAGAACUCUGAAC-/3'inverted dT/), and the products were enriched by a second round of streptavidin enrichment. Then, for 5' end repair, the RNA products were treated with RppH (Thermo Fisher) and polynucleotide kinase (PNK, NEB). The 5' repaired RNA was ligated to the reverse 5' RNA adaptor VRA5 (5'-CUGAACAAGCAGAAGACGGCAUACGA-3') before the third round of streptavidin bead enrichment. RNA was reverse-transcribed using RT primer (5'AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3'). The cDNA product was amplified for 18 cycles, and the library larger than 150 bp (insert > 70 bp) was purified from the PAGE gel. The library was quantified before NGS.