title: GSM1290725: child patient T3 (microcephaly), mutation CLP1 R140H; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA for sequencing was isolated from patient (BAB3401 and 3402) or parental (BAB3845 and 3846) fibroblasts using the TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions For each sample 300 ng of total RNA were hydrolyzed in a 15 μL buffer of 10 mM Na2CO3 and 10 mM NaHCO3 at ~pH 9.7, for 5 minutes at 90 °C.  The 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 °C for 1 h. The resulting RNA was re-phosphorylated with 10 U of T4 polynucleotide kinase (NEB) in a 20 µl reaction of 70 mM Tris-HCl pH 7.6 at 25 °C, 10 mM MgCl2, 5 mM DTT and 1 mM ATP, at 37 °C for 1 h. Fragments of 19-24 nts were converted into barcoded small RNA cDNA libraries and submitted for Illumina sequencing
attributes.tag: GEO Accession
attributes.value: GSM1290725