title: GSM5468775: ZfpKOTN-2; Mus musculus; RNA-Seq
library_construction_protocol: Approximately 1000 cells of desired type were lysed in 6 μl lysis buffer (0.2% Triton X-100 and 2 U/μl of RNase inhibitor). Lysates were mixed with 3 μl of anchored oligo-dT primer (10 μM, 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′) and 3 μl of dNTP mix (10 mM, Fermentas), denatured at 72 °C for 3 min and immediately placed on ice afterwards. The first-strand reaction mix containing 1 μl SuperScript II reverse transcriptase (Invitrogen), 0.5 μl RNAse inhibitor (Clontech), 4 μl Superscript II First-Strand Buffer (Invitrogen), 1 μl DTT (100 mM, Invitrogen), 4 μl betaine (5 M, Sigma), 0.12 μl MgCl2 (1 M, Sigma), 0.2 μl TSO (100 μM) and 0.58 μl nuclease-free water, were added to each sample. Reverse transcription reaction was carried out by incubating at 42 °C for 90 min, followed by 10 cycles of (50 °C for 2 min, 42 °C for 2 min). Finally, the reverse transcriptase was inactivated by incubation at 70 °C for 15 min. PCR mix containing 25 μl KAPA HiFi HotStart ReadyMix, 0.5 μl IS PCR primers (10 μM) and 1.1 μl nuclease-free water were added to each sample. The reaction was incubated at 98 °C for 3 min, then cycled 17 times (98 °C 20 s, 67 °C 15 s, 72 °C 6 min), with a final extension at 72 °C for 5 min. cDNAs were purified by Vazyme DNA clean beads following manufacturer's instructions. 1 ng of cDNA were used for the tagmentation reaction using Tn5 (Vazyme). The tagmentation reaction (20 μl) was incubated at 55 °C for 10 min and stopped by addition of 2 μl 0.2%SDS and incubated at 70 °C for 20 min. A second amplification was performed using Q5 polymerase (NEB) as follows: 72 °C 3 min, 98 °C 30 s, then 12 cycles of (98 °C 15 s, 60 °C 30 s, 72 °C 3 min), final extension at 72 °C 5 min. Libraries were purified using Vazyme DNA clean beads. smart-seq2