GSM4891292: MAC_3′seq_LPS 4h_rep2; Homo sapiens; OTHER
After washing the cells with DPBS three times, total RNA was extracted by directly adding TRIzol  to the plate with the adherent cells on it. 5 million cells were used for the construction of PRO-seq libraries. For 3′-seq and TED-seq, 10 ug of total RNAs were used. RNA libraries were prepared to be compatible with Illumina small RNA sequencing. We prepared 3′-seq libraries as follows: total RNAs was extracted with TRIzol (Invitrogen). For each sample, poly(A) RNAs were isolated from 10 ug total RNAs, and proceeded with RNA fragmentation, 5′ RNA phosphorylation, and 5′ RNA ligation. After heat denaturation at 65C for 2 min, 5′ adapter-ligated poly(A)-containing RNA fragments were reverse transcribed by superscript II enzyme (Invitrogen) using RT primer ( CPS_RTP: 5′ - GTTCAGAGTTCTACAGTCCGACGATC NNNNNNNNT8VN-3′) at 50 C for 1hr. CPS_RTP was designed to anneal to the junction of 3′ cleaved site and poly(A) tail using its short oligo dT residues and two degenerate nucleotides, and also to contain UMI barcode and adaptor for PCR deduplication, and universal PCR amplification, respectively. The resulting cDNA molecules were amplified by PCR, followed by gel purification of 200 to 500 bp PCR products on 6% PAGE gel in TBE buffer. The eluted PCR products proceed with sequencing on Illumina Nextseq 500.