GSM1521786: strand specific RNA-Seq for LRG derived astrocyte like populations replicate 2; Homo sapiens; RNA-Seq
RNA-Seq:RNA was extracted using the miRNeasy kit (Qiagen, 217004). Poly(A) RNA was isolated using Oligo d (T25) beads (NEB, E7490L). RNA-Seq: The following steps of Illumina library construction (end-repair, addition of A-base, ligation of barcoded adaptors and PCR enrichment) we used a general SPRI cleanup procedure: addition of PEG buffer (20% PEG and 2.5 M NaCl), and extracted and washed as above. The enzymatic reactions were carried as follows: 1. DNA end-repair: T4 PNK and T4 polymerase (New England Biolabs) incubated 12C for 15 min, 25C for 15 min;  2. A-base addition: Klenow (3’->5’ exonuclease; New England Biolabs) incubated at 37C for 30 min. 3. Adaptor ligation: DNA ligase (New England Biolabs) and indexed oligo adaptors and incubated 25C for 15 min, followed by 0.7X SPRI/reaction to remove non-ligated adaptors. 4. PCR enrichment: PCR mastermix (primer set, dNTP mix, Pfu Ultra Buffer (Agilent), Pfu Ultra-II Fusion (Agilent), water), for 20 cycles. The PCR amplified libraries we cleaned up using 0.7X SPRI/reaction (size selection mode) to remove excessive primers. Roughly 5 picomoles of DNA library was then applied to each lane of the flow cell and sequenced on Illumina HiSeq 2000 sequencers according to standard Illumina protocols.