title: GSM2399972: NM_seq_HeLa_total_RNA_rep2; Homo sapiens; OTHER
library_construction_protocol: Total RNA was purified from the cells using 5PRIME PerfectPure RNA Cultured Cell kit (cat#2302340) and polyA+ selection was carried out using GenElute mRNA miniprep kit (Sigma Aldrich, cat#MRN70). RNA was fragmented chemically to ~100 nts long fragments and subjected to IP using anti-m6A antibodies. 1st strand cDNA was synthesized using Illumina random primers and Invitrogen SuperScript II Reverse Transcriptase (Cat# 18044-014) then 2strand cDNA was synthesized by Illumina's mRNA Sample preparation Kit (Part# 1004814). DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation  ~200 bp DNA fragments (insert plus adaptor and PCR primer sequences)  were isolated from 2% agarose gel. Purified DNA fragments were PCR amplified with Illumina primers for 15 cycles and 10pM DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq2000 following the manufacturer's protocols. NEBNext Small RNA Library Prep Kit (cat.# E7300)
identifiers.namespace: GEO
identifiers.id: GSM2399972