GSM4568936: Ba14; Homo sapiens; ncRNA-Seq
Total RNA for sequencing was isolated using the TRIzol Reagent (Invitrogen) according to the manufacturer's instructions For hydro-tRNAseq: 4 μg of 60-100 nt-sized total RNA were hydrolyzed in a 15 μL buffer of 10 mM in a 15 µL buffer of 10 mM Na2CO3 and 10 mM NaHCO3 at pH 9.7 at 65 °C for 1 h. The partially hydrolyzed RNA was dephosphorylated with 10 U of calf intestinal phosphatase (NEB) in a 50 µL reaction of 100 mM NaCl, 50 mM Tris-HCl, pH 7.9 at 25 °C, 10 mM MgCl2, 1 mM DTT, 3 mM Na2CO3 and 3 mM NaHCO3, at 37 ºC for 1 h. The resulting RNA was re-phosphorylated with 10 U of T4 polynucleotide kinase (NEB) in a 20 µlL reaction of 70 mM Tris-HCl, pH 7.6, 10 mM MgCl­2, 5 mM DTT and 1 mM ATP, at 37 °C for 1 h. Fragments of 19-35 nt were converted into barcoded small RNA cDNA libraries. Samples Ba12, Ba13, Ba17, Ba19, Ba245 were part of a barcoded library in a single lane. 3' adapters used (frist 5 nts represent barcodes): Ba12  TCCTATCGTATGCCGTCTTCTGCTTG, Ba13, TCATCTCGTATGCCGTCTTCTGCTTG, Ba17  TCCACTCGTATGCCGTCTTCTGCTTG, Ba19, TCGATTCGTATGCCGTCTTCTGCTTG, Ba245 TCTAGTCGTATGCCGTCTTCTGCTTG, 5' adapter (common): AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACGATC. Samples Ba1230, Ba14, Ba1597, Ba1613, Ba1614 were part of a second barcoded library in a single lane. 3' adapters used (first 5 nts represnt barocdes): Ba1230, TCTAGTCGTATGCCGTCTTCTGCTTG, Ba14  TCGATTCGTATGCCGTCTTCTGCTTG, Ba1597 TCCTATCGTATGCCGTCTTCTGCTTG, Ba1613  TCCACTCGTATGCCGTCTTCTGCTTG, Ba1614  TCATCTCGTATGCCGTCTTCTGCTTG, 5' adapter (common): AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACGATC. Two size markers were used as controls: 19-nt marker: CGTACGCGGGTTTAAACGA; 35-nt marker: TCATCTTGGTCGTACGCGGAATAGTTTAAACTGT
GEO Accession
GSM4568936