Biotin t15 tt 5

The 3′READS procedure (3′READS+ version) was described in ref. ^{40 (link)}. Briefly, Poly(A)+ RNA in 0.1 or 1 µg of input RNA was captured using oligo(dT)₂₅ magnetic beads (NEB) and fragmented on the beads using RNase III (NEB). After washing away unbound RNA fragments, poly(A)+ RNA fragments were eluted from the beads and precipitated with ethanol, followed by ligation to heat-denatured 5′ adapter (5′-CCUUGGCACCCGAGAAUUCCANNNN) with T4 RNA ligase 1 (NEB). The ligation products were captured by biotin-T₁₅-(+TT)₅ (Exiqon) bound to Dynabeads MyOne Streptavidin C1 (Thermo Fisher). After washing, RNA fragments on the beads were incubated with RNase H to remove bulk of the poly(A) tail and then eluted from the beads. After precipitation with ethanol, RNA fragments were ligated to a 5′ adenylated 3′ adapter (5′-rApp/NNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC (Bioo Scientific) with T4 RNA ligase 2 (truncated KQ version, NEB). The ligation products were then precipitated and reverse transcribed using M-MLV reverse transcriptase (Promega), followed by PCR amplification using Phusion high-fidelity DNA polymerase (NEB) and bar-coded PCR primers for 15–18 cycles. PCR products were size-selected twice with AMPure XP beads (Beckman Coulter). The size and quantity of the libraries were examined on an Agilent Bioanalyzer. Libraries were sequenced on an Illumina NextSeq 500 (1 × 75 bases).

The 3′READS+ procedure was carried out as previously described (Zheng et al., 2016 (link)). Briefly, poly(A)+ RNA was captured by using oligo (dT)₂₅ magnetic beads (NEB) and was fragmented on-bead by RNase III (NEB). After washing away free RNA fragments, poly(A)+ RNA fragments were eluted from the beads and precipitated with ethanol, followed by ligation to heat-denatured 5′ adapter (5′-CCUUGGCACCCGAGAAUUCCANNNN) with T4 RNA ligase 1 (NEB). The ligated products were captured by biotin-T₁₅-(+TT)₅ (Exiqon, and +T indicates locked nucleic acid) bound to Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). After washing, RNA fragments on the beads were digested with RNase H and then eluted from the beads. After precipitation with ethanol, RNA fragments were ligated to a 5′ adenylated 3′ adapter (5′-rApp/NNNGATCGTCGGACTGTAGAACTCTGAAC/3ddC (Bioo Scientific) with T4 RNA ligase 2 (truncated KQ version, NEB). The ligation products were then reverse transcribed by using M-MLV reverse transcriptase (Promega), followed by PCR amplification with Phusion high-fidelity DNA polymerase (NEB) and bar-coded PCR primers for 12–18 cycles. PCR products were size selected twice with AMPure XP beads (Beckman Coulter). The size and quantity of the cDNA libraries were examined on an Agilent Bioanalyzer and sequenced on an Illumina HiSeq machine (1 × 150 bases).