Library prep using Zymo SwitchFree kit on spat samples with PolyA isolation and pooling - 111625

Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit for in-house library prep of coral spat samples treated for PolyA isolation and pooling - 111625

Protocols used

Using the Zymo-Seq SwitchFree 3′ mRNA Library Kit (CAT. R3009-A, LOT. 252649) to prepare libraries starting from RNA samples exctracted from coral spat of Montipora capitata. RNA extraction posts can be found here, here and here. NEB PolyA treatment post can be found here.

The idea is to use the NEB kit before performing the library prep to reduce the amount of rRNA present in the libraries.

For the library prep I followed the protocol of Jill Ashey and the kit protocol above. I’m using 1.05 ng of RNA input.

Here I’m using UDI primers from the 96 well plate (CAT. D3096, LOT. 250838) provided with the kit.

All samples were eluted in 15uL of elution buffer.

Library prep

I ran the library prep protocol linked above. I used 23 PCR cycles. With 15 samples, it took about 7 hours to run the protocol and do QC on the final library.

Here’s a breakdown of input RNA volume and quantity:

sample_id   RNA_QBIT_AVG (ng/uL)   RNA (uL)   Ultrapure water (uL)   Total input RNA (ng)
B8   0.4835   2.17   2.83   1.05
C10   0.431   2.44   2.56   1.05
D1   0.32   3.28   1.72   1.05
A4   0.255   4.12   0.88   1.05
A9   0.514   2.04   2.96   1.05
D8   0.5635   1.86   3.14   1.05
C9   0.3315   3.17   1.83   1.05
C13   0.275   3.82   1.18   1.05
B6   0.33   3.18   1.82   1.05
B11   0.463   2.27   2.73   1.05
D6   0.2975   3.53   1.47   1.05
D11   0.211   4.98   0.02   1.05
A6   0.45   2.33   2.67   1.05
C6   0.4735   2.22   2.78   1.05
D12   0.624   1.68   3.32   1.05

Qubit Results

I used High Sensitivity dsDNA Qubit Protocol linked above. Library samples were read twice, standard only read once.

  • Standard 1: 50.82
  • Standard 2: 22722.93
QBIT date   sample_id   Species   Library read1   Library read2   Library_AVG (ng/ul)
20251116   B8   Montipora capitata   LOW   LOW   LOW
20251116   C10   Montipora capitata   3.98   3.88   3.93
20251116   D1   Montipora capitata   4.58   4.48   4.53
20251116   A4   Montipora capitata   5.08   5.00   5.04
20251116   A9   Montipora capitata   9.88   9.66   9.77
20251116   D8   Montipora capitata   9.30   9.18   9.24
20251116   C9   Montipora capitata   3.16   3.10   3.13
20251116   C13   Montipora capitata   12.5   12.0   12.25
20251116   B6   Montipora capitata   1.75   1.76   1.755
20251116   B11   Montipora capitata   14.1   13.9   14.0
20251116   D6   Montipora capitata   18.9   18.7   18.8
20251116   D11   Montipora capitata   21.8   21.2   21.5
20251116   A6   Montipora capitata   14.7   14.6   14.65
20251116   C6   Montipora capitata   12.2   12.0   12.1
20251116   D12   Montipora capitata   15.5   15.2   15.35

TapeStation

After the library prep, I run samples on the tapestation (HS D1000), protocol linked above. Full tapestation report here.

TapeStation date   sample_id   TapeStation conc. pg/uL   Primer set
20251116   B8   2.59   17
20251116   C10   2410   18
20251116   D1   2320   19
20251116   A4   293   20
20251116   A9   4500   21
20251116   D8   /   22
20251116   C9   1520   23
20251116   C13   4890   24
20251116   B6   424   25
20251116   B11   6610   26
20251116   D6   7000   27
20251116   D11   8660   28
20251116   A6   7640   29
20251116   C6   5730   30
20251116   D12   7130   31

tapeS.png

  • It is weird that D8 has 9.24ng/ul from the qbit and nothing from the tapestation, I re-ran this sample and it worked.

tapeS2.png

Libraries pooling

Pooling calculations were done following Zoe’s protocol.

Library_prep_date   Species   Sample.ID   Library_ID   Primer   TubeID   Average_Qubit_Conc   TS_Peak_Size   Molarity_nM_Qubit   Amount_Library_uL   Amount_Buffer_uL  
20251116   Mcap   C10   C10   18   C10   3.93   514   11.58   2.59   7.41  
20251116   Mcap   D1   D1   19   D1   4.53   518   13.25   2.26   7.74  
20251116   Mcap   A4   A4   20   A4   5.04   513   14.89   2.02   7.98  
20251116   Mcap   A9   A9   21   A9   9.77   399   37.1   0.81   9.19  
20251116   Mcap   D8   D8   22   D8   9.24   447   31.32   0.96   9.04  
20251116   Mcap   C9   C9   23   C9   3.13   458   10.35   2.9   7.1  
20251116   Mcap   C13   C13   24   C13   12.25   453   40.97   0.73   9.27  
20251116   Mcap   B6   B6   25   B6   1.755   496   5.36   5.6   4.4  
20251116   Mcap   B11   B11   26   B11   14   437   48.54   0.62   9.38  
20251116   Mcap   D6   D6   27   D6   18.8   426   66.87   0.45   9.55  
20251116   Mcap   D11   D11   28   D11   21.5   402   81.03   0.37   9.63  
20251116   Mcap   A6   A6   29   A6   14.65   389   57.06   0.53   9.47  
20251116   Mcap   C6   C6   30   C6   12.1   425   43.14   0.7   9.3  
20251116   Mcap   D12   D12   31   D12   15.35   399   58.29   0.51   9.49  
  • I normalized the libraries into 1 pool with 4nM concentration. Total volume for pool 1 is 140uL, I will send only 35uL (the minumum volume they need is 30uL).
  • For each library, I added calcucated amount of DNA elution buffer (“Amount_H20”) and the library (“Amount_Library”) to a new PCR tube and mixed well
  • I then combined all of these tubes into one LoBind 1.5 mL tube
  • I quantified the pooled libraries with Qubit dsDNA HS Assay Kit and tapestation analysis with the HS D1000 ScreenTape. Then froze until shipping.
Final pool, sent on 11/18/25 to Oklahoma

Qubit Results

I used High Sensitivity dsDNA Qubit Protocol linked above. Library sample was read twice, standard only read once.

  • Standard 1: 73.95
  • Standard 2: 26285.98
QBIT date   sample_id   Library read1   Library read2   Library_AVG (ng/ul)
20251117   POOL_F   0.878   0.844   0.861

TapeStation

After pooling, I ran the library on the tapestation (D1000 HS), protocol linked above. Full tapestation report here

Written on November 16, 2025